U.S. patent application number 17/499265 was filed with the patent office on 2022-09-01 for methods for assembling and reading nucleic acid sequences from mixed populations.
The applicant listed for this patent is METABIOTECH CORPORATION. Invention is credited to James A. STAPLETON, Timothy WHITEHEAD.
Application Number | 20220275437 17/499265 |
Document ID | / |
Family ID | 1000006333657 |
Filed Date | 2022-09-01 |
United States Patent
Application |
20220275437 |
Kind Code |
A1 |
STAPLETON; James A. ; et
al. |
September 1, 2022 |
METHODS FOR ASSEMBLING AND READING NUCLEIC ACID SEQUENCES FROM
MIXED POPULATIONS
Abstract
The disclosure relates to methods for obtaining nucleic acid
sequence information by constructing a nucleic acid library and
reconstructing longer nucleic acid sequences by assembling a series
of shorter nucleic acid sequences.
Inventors: |
STAPLETON; James A.;
(Eugene, OR) ; WHITEHEAD; Timothy; (East Grand
Rapids, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
METABIOTECH CORPORATION |
San Jose |
CA |
US |
|
|
Family ID: |
1000006333657 |
Appl. No.: |
17/499265 |
Filed: |
October 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17171672 |
Feb 9, 2021 |
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17499265 |
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16253898 |
Jan 22, 2019 |
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17171672 |
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14947988 |
Nov 20, 2015 |
10233490 |
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16253898 |
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62083057 |
Nov 21, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6806 20130101 |
International
Class: |
C12Q 1/6869 20060101
C12Q001/6869; C12Q 1/6806 20060101 C12Q001/6806 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under
GM099291 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for sequencing a nucleic acid molecule, the method
comprising: (a) providing a plurality of clonal nucleic acid
molecules each comprising at least a first adapter having the same
barcode sequence attached in proximity to a first end; (b)
fragmenting the clonal nucleic acid molecules adjacent to random
portions of the nucleic acid molecules to provide a second end; (c)
joining the first end with the second end to provide a plurality of
circularized nucleic acid molecules having the barcode sequence
adjacent to the random portion of the nucleic acid molecule; (d)
fragmenting the plurality of circularized nucleic acid molecules
from (c); (e) sequencing the barcode and the random portions of the
nucleic acid molecules; and (f) assembling the sequences from the
plurality of random portions of the nucleic acid molecules.
2. The method of claim 1, wherein the method is performed with a
plurality of clonal nucleic acid populations, each population
having a different barcode sequence attached thereto, and a
separate sequence is assembled in (f) for each of the barcode
sequences.
3. (canceled)
4. The method of claim 2, comprising, prior to step (a): (i)
providing a plurality of nucleic acid molecules; (ii) providing a
plurality of first adapters, each adapter comprising a first region
that is identical for each of the adapters and a barcode sequence
that is unique for each of the adapters; (iii) attaching the
adapters of (ii) to the target nucleic acid molecules of (i) to
create a plurality of target molecules with adapters at both the 5'
and 3' ends; and (iv) amplifying the plurality of nucleic acid
molecules molecules of (iii) to produce the plurality of clonal
nucleic acid populations, wherein the nucleic acid molecules in
each population having the same barcode sequence attached in
proximity to the first end.
5. The method of claim 4, wherein the attaching in (iii) is
performed by PCR.
6. The method of claim 4, wherein the attaching in (iii) is
performed by ligation.
7. The method of claim 1: wherein the first adapter comprises an
outer polymerase chain reaction (PCR) primer region or nucleic acid
amplification region and an inner sequencing primer region, and the
barcode sequence.
8. The method of claim 4, further comprising fragmenting a nucleic
acid molecule comprising a target nucleotide sequence into a
plurality of linear nucleic acid molecules prior to attaching the
first adapter.
9. The method of claim 4, wherein the nucleic acid molecules
comprise first adapters attached at the 5' and the 3' ends, and
wherein the first adapter attached at the 5' end comprises a
different barcode sequence than the first adapter attached at the
3' end.
10. The method of claim 4, wherein the nucleic acid molecules
comprise first adapters attached at the 5' and the 3' ends, and
wherein the first adapter attached at the 5' end and the first
adapter attached at the 3' end comprise the same barcode
sequence.
11. The method of claim 4, wherein amplifying the plurality of
target molecules comprises PCR.
12. The method of claim 11, wherein amplifying the plurality of
nucleic acid molecules is carried out using a primer complementary
to the PCR primer region.
13. The method of claim 11, further comprising removing the PCR
primer region from the plurality of nucleic acid molecules after
amplification.
14. The method of claim 13, wherein the removing the PCR primer
region is carried out before joining the first end with the second
end to provide a plurality of circularized nucleic acid
molecules.
15. The method of claim 1, wherein fragmenting the plurality of
clonal nucleic acid molecules at (b) is carried out by enzymatic,
chemical or physical shearing.
16. The method of any one of claim 1, wherein fragmenting the
plurality of clonal nucleic acid molecules at (b) is carried out at
random locations on the nucleic acid sequences.
17. The method of claim 83, wherein the second adapter comprises
two nucleic acid strands of different lengths, wherein the strand
attached at the 5' end of a fragmented nucleic acid molecule is of
a different length than the strand attached at the 3' end of the
fragmented nucleic acid molecule, wherein one end of the second
adapter is double stranded to facilitate ligation and the other end
of the second adapter comprises a 3' single-stranded overhang, and
wherein only the longer of the two oligonucleotides comprises a
sequence complementary to a second sequencing primer and comprises
sufficient length to allow annealing of that primer.
18. The method of claim 83, wherein replicating the nucleic acid
molecules comprising the first and second adapters is carried out
using two primers, the first of which is complementary to a
constant sequence from the first adapter, and the second of which
is complementary to an overhanging sequence of the second adapter,
wherein the second adapter is an asymmetric adaptor, and wherein
both adapters together add sequences necessary for nucleic acid
sequencing.
19. The method of claim 18, wherein the replicating is carried out
using PCR.
20. (canceled)
21. The method of claim 2, wherein sequences from the plurality of
clonal nucleic acid populations are sorted into independent groups
is based on shared barcodes.
22. The method of claim 21, wherein assembling each group is
carried out independent of all other groups.
23. The method of claim 4, further comprising selecting the
plurality of nucleic acid molecules on the basis of size prior to
attaching the first adapter.
24. The method of claim 1, further comprising selecting the nucleic
acid molecules on the basis of size prior to sequencing.
25. The method of claim 1, wherein fragmenting the plurality of
circularized nucleic acid molecules at step (d) comprises
enzymatic, chemical or physical shearing.
26. The method of claim 13, wherein the PCR primer region is
removed by an enzyme that excises uracils and breaks the phosphate
backbone.
27. The method of claim 13, wherein the PCR primer region comprises
methylated nucleotides and the PCR primer region is removed by
restriction enzymes specific for methylated sequences.
28. The method of claim 1, wherein nucleic acid sequence
information is obtained for a longer nucleic acid sequence
comprising a length of at least about 500 bases or at least about
1000 bases.
29-30. (canceled)
31. The method of claim 1, wherein nucleic acid sequence
information is obtained for a longer nucleic acid sequence
comprising a length from about 1 kilobase to about 20
kilobases.
32. (canceled)
33. The method of claim 1, wherein the nucleic acid sequence
information comprises greater than about 95% fidelity to the target
nucleotide sequence.
34. The method of claim 1, wherein the target nucleotide sequence
originates from genomic DNA.
35-82. (canceled)
83. The method of claim 1, further comprising after step (d) and
before step (e): (i) attaching a second adapter to both ends of the
plurality of fragmented nucleic acid molecules; and (ii)
replicating all or part of the plurality of nucleic acid
molecules.
84. The method of claim 15, wherein the physical shearing comprises
acoustic shearing, sonication, or hydrodynamic shear.
85. The method of claim 15, wherein the enzymatic shearing
comprises a fragmentase, KAPA Frag Enzyme, DNase I, a non-specific
nuclease, a transposase, a restriction endonuclease, or a
tagmentase.
86. The method of claim 15, wherein the enzymatic shearing
comprises a double-stranded DNA fragmentase.
87. The method of claim 25, wherein the physical shearing comprises
acoustic shearing, sonication, or hydrodynamic shear.
88. The method of claim 25, wherein the enzymatic shearing
comprises a fragmentase, KAPA Frag Enzyme, DNase I, a non-specific
nuclease, a transposase, a restriction endonuclease, or a
tagmentase.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 17/171,672, filed Feb. 9, 2021, which is a continuation of U.S.
application Ser. No. 16/253,898, filed Jan. 22, 2019, which is a
continuation of U.S. application Ser. No. 14/947,988, filed Nov.
20, 2015, now U.S. Pat. No. 10,233,490, which claims the benefit of
U.S. Provisional Patent Application Ser. No. 62/083,057, filed Nov.
21, 2014, each of which is incorporated herein by reference in its
entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 4, 2016, is named 46679701201_SL.txt and is 14,291 bytes in
size.
BACKGROUND
[0004] The transition from traditional Sanger-style sequencing
methods to next-generation sequencing methods has lowered the cost
of sequencing, yet significant limitations of next-generation
sequencing methods remain. In one respect, available sequencing
platforms generate sequencing reads that, while numerous, are
relatively short and can require computational reassembly into full
sequences of interest. Available assembly methods can be slow,
laborious, expensive, computationally demanding, and/or unsuitable
for populations of similar individuals (e.g., viruses). This is
especially true for sequencing of complex genomes. Assembly is
challenging, in part due to the ever-swelling sequencing datasets
associated with assembly of short reads. Such datasets can place a
large strain on computer clusters. For example, de novo assembly
can require that sequencing reads (or k-mers derived from them) be
stored in random access memory (RAM) simultaneously. For large
datasets this requirement is not trivial. Moreover, even when
assembly is possible, crucial haplotype information often cannot be
recovered. Indeed, inherent limitations of available technologies
obstruct improvements to overcoming the shortcomings of status quo
sequencing technologies. Thus, there exists a need for improved
sequencing methods and associated assembly techniques that reduce
the time and/or computational requirements necessary to obtain
accurate sequences.
SUMMARY
[0005] The disclosure provides a new method for obtaining nucleic
acid sequence information. In various aspects, the method permits
the sequencing of target nucleic acids by assembling intermediate
and long nucleic acid sequences from short nucleic acid sequences.
In various aspects, the method solves a problem associated with
current nucleic acid sequencing methods, wherein important
information about the origin of each short nucleic acid sequence is
lost. In various aspects, the method is useful in haplotyping
because it allows for identification and differentiation of
mutations on the same or different chromosomes. In various aspects,
the method is quicker and more accurate than other methods for
obtaining nucleic acid sequence information.
[0006] In some aspects, the disclosure relates to methods for
obtaining nucleic acid sequence information from a nucleic acid
molecule by assembling a series of short nucleic acid sequences
into longer nucleic acid sequences.
[0007] In one embodiment described herein is a method for obtaining
nucleic acid sequence information from a nucleic acid molecule by
assembling a series of nucleic acid sequences into a (i.e., one or
more) longer nucleic acid sequence. In some aspects, the longer
nucleic acid sequence or sequences are intermediate or long nucleic
acid sequences. The methods described here allow subsets of the
larger number of short reads collected during a sequencing run to
be independently assembled into the full sequence of the
intermediate-length molecule from which the "barcoded" group of
short reads derived. These "sub-assembled" sequences are referred
to as "synthetic long reads," because while like a true long read
they represent the contiguous sequence of a particular individual
nucleic acid molecule, they are synthesized from a collection of
short reads that are grouped by a shared "barcode" sequence
tag.
[0008] In one embodiment, the disclosure provides a method for
obtaining nucleic acid sequence information from a nucleic acid
molecule comprising a target nucleotide sequence by assembling a
series of nucleic acid sequences into a (one or more) longer
nucleic acid sequences, said method comprising: attaching a first
adapter comprising an outer polymerase chain reaction (PCR) primer
region, an inner sequencing primer region, and a central barcode
region to each end of a plurality of linear nucleic acid molecules
to form barcode-tagged molecules; replicating the barcode-tagged
molecules to obtain a library of barcode-tagged nucleic acid
molecules; breaking the barcode-tagged molecules, thereby
generating linear, barcode-tagged fragments comprising the barcode
region at one end and a region of unknown sequence at the other
end; circularizing the linear, barcode-tagged fragments comprising
the barcode region at one end and a region of unknown sequence from
an interior portion of the target nucleotide sequence at the other
end, thereby bringing the barcode region into proximity with the
region of unknown sequence; fragmenting the circularized,
barcode-tagged fragments into linear, barcode-tagged fragments;
attaching a second adapter to each end of the linear,
barcode-tagged fragments to form double adapter-ligated
barcode-tagged nucleic acid fragments; replicating all or part of
the double adapter-ligated barcode-tagged nucleic acid fragments;
sequencing the double adapter-ligated barcode-tagged nucleic acid
fragments; sorting a series of sequenced nucleic acid fragments
into independent groups; and assembling each group of reads or
short reads into a (one or more) longer nucleic acid sequence.
[0009] In some aspects, the method further comprises fragmenting a
nucleic acid molecule into a plurality of shorter, linear nucleic
acid sequences comprising target nucleotide sequences prior to
attaching the first adapter. Such fragmenting is necessary, in some
aspects, when the nucleic acid molecule is genomic DNA.
[0010] In some aspects of the method, the first adapter attached at
the 5' end comprises a different barcode than the first adapter
attached at the 3' end. In some aspects, the first adapter attached
at the 5' end and the first adapter attached at the 3' end
comprises the same barcode.
[0011] In some aspects of the method, the barcode-tagged sequences
are replicated to obtain many copies of each barcode-tagged
sequence. In some aspects, the replication is carried out using a
primer complementary to the PCR primer region. In some aspects, the
replication, or nucleic acid amplification, is carried out by any
suitable nucleic acid amplification method. In various aspects,
nucleic acid amplification methods include, but are not limited to,
PCR, quantitative PCR, quantitative fluorescent PCR (QF-PCR),
multiplex fluorescent PCR (MF-PCR), real time PCR (RTPCR), single
cell PCR, restriction fragment length polymorphism PCR (PCR-RFLP),
PCK-RFLPIRT-PCR-IRFLP, hot start PCR, nested PCR, in situ polony
PCR, in situ rolling circle amplification (RCA), bridge PCR,
picotiter PCR, and emulsion PCR. Other suitable amplification
methods include, but are not limited to, ligase chain reaction
(LCR), transcription amplification, self-sustained sequence
replication, selective amplification of target nucleic acids,
consensus sequence primed polymerase chain reaction (CP-PCR),
arbitrarily primed polymerase chain reaction (AP-PCR), degenerate
oligonucleotide-primed PCR (DOP-PCR), and nucleic acid-based
sequence amplification (NABSA). In particular aspects, the nucleic
acid amplification is carried out by PCR.
[0012] In some aspects, the method further comprises of removing
the PCR primer region from the barcode-tagged sequences. In some
aspects, removing the PCR primer region is carried out before the
circularizing the barcode-tagged fragments. In some aspects,
removing the PCR primer region is carried out before breaking the
barcode-tagged sequences at random locations. In some aspects,
removing the PCR primer region is carried out after breaking the
barcode-tagged sequences at random locations.
[0013] In some aspects of the method, breaking the barcode-tagged
sequences is carried out by an enzyme. In some aspects, the
breaking is carried out at random locations on the nucleic acid
sequences.
[0014] In some aspects, the second adapter comprises, consists
essentially of, or consists of two annealed nucleic acid strands of
different lengths, wherein the strand attached at the 5' ends of a
linear, barcode-tagged fragment is of a different length than the
strand attached at the 3' ends of a linear, barcode-tagged
fragment, wherein one end of the second adapter is double stranded
to facilitate ligation and the other end of the second adapter
comprises a 3' single-stranded overhang, and wherein only the
longer of the two oligonucleotides comprises a sequence
complementary to a second sequencing primer and comprises
sufficient length to allow annealing of that primer.
[0015] In some aspects, replicating the double adapter-ligated
barcode-tagged nucleic acid fragments is carried out using two
primers, the first of which is complementary to a constant sequence
from the barcode-containing adapter, and the second of which is
complementary to the overhanging sequence of the asymmetric
adapter, and which together add sequences necessary for nucleic
acid sequencing.
[0016] In some aspects, sequencing the double adapter-ligated
barcode-tagged nucleic acid fragments is carried out beginning with
the barcode region followed by the target sequence.
[0017] In some aspects of the method, sorting the series of
sequenced nucleic acid fragments into independent groups is based
on shared barcodes.
[0018] In some aspects, assembling each group is carried out
independent of all other groups.
[0019] In various aspects, the method further comprises a (one or
more) selecting the plurality of linear nucleic acid sequences on
the basis of size. In some aspects, this selecting by size is
carried out prior to attaching the first adapter or after attaching
the first adapter but prior to nucleic acid amplification. In some
aspects, selecting the fragments on the basis of size is carried
out prior to sequencing.
[0020] In some aspects, the enzyme or enzyme mixture that breaks
the linear, tagged nucleic acid fragments is double-stranded DNA
fragmentase. In some aspects, the enzyme or enzyme mixture that
breaks the linear, tagged nucleic acid fragments is KAPA Frag
Enzyme. In some aspects, the enzyme or enzyme mixture that breaks
the linear, tagged nucleic acid fragments is a transposase.
[0021] In various aspects of the method, the PCR primer region is
removed from the tagged, amplified nucleic acid fragments. In some
aspects, the PCR primer region is removed by an enzyme or enzyme
mixture that excises uracils and breaks the phosphate backbone. In
some aspects, the PCR primer comprises methylated nucleotides and
the PCR primer region is removed by restriction enzymes specific
for methylated sequences. In some aspects, all or part of the PCR
primer comprises RNA and the primer region is removed by enzymes
specific for RNA.
[0022] In some aspects, nucleic acid sequence information is
obtained for a longer nucleic acid sequence comprising a length of
at least about 500 bases. In some aspects, nucleic acid sequence
information is obtained for an intermediate or a long nucleic acid
sequence. In some aspects, nucleic acid sequence information is
obtained for a nucleic acid sequence comprising a length of at
least about 1000 bases. In some aspects, nucleic acid sequence
information is obtained for a nucleic acid sequence comprising a
length of at least 1000 or more bases. In some aspects, nucleic
acid sequence information is obtained for a nucleic acid sequence
comprising a length from about 1 kilobase to about 20 kilobases. In
some aspects, nucleic acid sequence information is obtained for a
nucleic acid sequence comprising a length of up to about 12
kilobases.
[0023] In some aspects, the nucleic acid sequence information
comprises greater than about 95% fidelity to the target nucleotide
sequence.
[0024] In some aspects, the target nucleotide sequence originates
from genomic DNA. In some aspects, the target nucleotide sequence
is a non-genomic nucleic acid.
[0025] In some aspects, the nucleic acid sequence information is
obtained in less than three days.
[0026] In some aspects, the method is carried out in a single tube.
In some aspects, samples for barcode pairing are prepared in
parallel in a second tube.
[0027] An additional aspect of the disclosure provides a method for
sequencing a nucleic acid molecule. The method includes: (a)
providing a plurality of clonal nucleic acid molecules each having
the same barcode sequence attached in proximity to a first end; (b)
for each nucleic acid molecule, fragmenting the nucleic acid
molecule adjacent to a random portion of the nucleic acid molecule
to provide a second end; (c) for each nucleic acid molecule,
joining the first end with the second end to provide a circularized
nucleic acid molecule having the barcode sequence adjacent to the
random portion of the nucleic acid sequence; (d) for each nucleic
acid molecule, sequencing the barcode and the random portion of the
nucleic acid molecule; and (e) assembling the sequence of the
nucleic acid molecule from the plurality of random portions of the
nucleic acid molecule. In some embodiments, the method is performed
with a plurality of clonal nucleic acid populations each having a
different barcode sequence attached thereto, and a separate
sequence is assembled in (e) for each of the barcode sequences.
[0028] An additional aspect of the disclosure provides a method
that comprises: (a) providing a plurality of target nucleic acid
molecules; (b) providing a plurality of adapter fragments, each
comprising a first region that is identical for each of the adapter
fragments and a second region that is unique for each of the
adapter fragments; (c) attaching the adapter fragments of (b) to
the target nucleic acid molecules of (a) to create a plurality of
adapter-ligated target molecules; (d) amplifying the
adapter-ligated target molecules of (c); (e) fragmenting the
amplified molecules of (d); (f) circularizing the fragmented
molecules of (e); (g) fragmenting the circularized molecules of
(f); and (h) sequencing the fragmented molecules of (g).
[0029] Another aspect of the disclosure provides a method that
comprises: (a) providing a plurality of target nucleic acid
molecules; (b) providing a plurality of adapter fragments, each
comprising a first region that is identical for each of the adapter
fragments and a second region that is unique for each of the
adapter fragments; (c) attaching the adapter fragments of (b) to
the target nucleic acid molecules of (a) to create a plurality of
adapter-ligated target molecules; (d); (e) amplifying the
adapter-ligated target molecules of (c); (f) fragmenting the
amplified molecules of (d); (g) circularizing the fragmented
molecules of (e); and (h) sequencing the circularized molecules of
(f).
[0030] In some aspects, the attaching in (c) is performed by
polymerase chain reaction (PCR). In some aspects, the attaching in
(c) is performed by ligation.
[0031] Another aspect of the disclosure provides a method
comprising: (a) sequencing a plurality of nucleic acids located at
positions on an array; and (b) measuring a phenotype of a molecule
at the positions on the array.
[0032] An additional aspect of the disclosure provides a method
comprising sequencing a genetic component of the members of a
polypeptide display library.
[0033] An additional aspect of the disclosure provides a method for
generating a plurality of linked sequence-phenotype pairs, the
method comprising: (a) applying to an array, a library of mutant
proteins associated with their encoding nucleic acid, wherein the
library is applied with essentially one mutant per array position;
(b) measuring the phenotype of the protein at each array position;
and (c) sequencing at least part of the nucleic acid associated
with the protein at each array position, thereby generating a
linked sequence-phenotype pair at each array position.
[0034] An additional aspect of the disclosure provides a method for
generating a plurality of linked sequence-phenotype pairs, the
method comprising: (a) applying to an array, a library of mutant
nucleic acids, wherein the library is applied with essentially one
mutant per array position; (b) measuring the phenotype of the
nucleic acid at each array position; and (c) sequencing at least
part of the nucleic acid at each array position, thereby generating
a linked sequence-phenotype pair at each array position.
[0035] An additional aspect of the disclosure provides a method for
generating a plurality of linked sequence-phenotype pairs, the
method comprising: (a) applying to an array, a library of mutant
nucleic acids, wherein the library is applied with essentially one
mutant per array position; (b) expressing the proteins encoded by
the nucleic acids on the array; (c) measuring the phenotype of the
proteins at each array position; and (d) sequencing at least part
of the nucleic acid at each array position, thereby generating a
linked sequence-phenotype pair at each array position.
[0036] An additional aspect of the disclosure provides a method for
generating a plurality of linked sequence-phenotype pairs, the
method comprising: (a) synthesizing a plurality of nucleic acids at
fixed positions on an array; (b) expressing the proteins encoded by
the nucleic acids on the array; and (c) measuring the phenotype of
the protein at each array position, thereby generating a linked
sequence-phenotype pair at each array position.
[0037] An additional aspect of the disclosure provides a method for
generating a plurality of linked sequence-phenotype pairs, the
method comprising: (a) applying to an array of immobilized nucleic
acids, a library of mutant proteins associated with their encoding
nucleic acid, wherein the immobilized nucleic acids hybridize with
the nucleic acids that are associated with the mutant proteins; and
(b) measuring the phenotype of the protein at each array position,
thereby generating a linked sequence-phenotype pair at each array
position.
[0038] In some aspects, the method further comprises analyzing the
linked sequence-phenotype pairs to determine: (i) a sequence that
expresses or has a high probability of expressing a protein having
a desired phenotype; and/or (ii) a plurality of sequences, wherein
at least one of the sequences has a high probability of expressing
a protein having a desired phenotype; and/or (iii) the effect of
individual sequence mutations on the phenotype of the protein
expressed from the sequence; and/or (iv) the effect of a group of
sequence mutations on the phenotype of the protein expressed from
the sequence; and/or (v) a set of allowed mutations at a sequence
position, wherein the protein expressed from the sequence has an
acceptable phenotype.
[0039] In some aspects, the method further comprises analyzing the
linked sequence-phenotype pairs to determine: (1) a nucleic acid
molecule that has a high probability of having a desired phenotype;
and/or (2) a plurality of nucleic acid molecules, wherein at least
one of the molecules that has a high probability of having a
desired phenotype; and/or (3) the effect of individual sequence
mutations on the phenotype of a nucleic acid molecule; and/or (4)
the effect of a group of sequence mutations on the phenotype of a
nucleic acid molecule; and/or (5) a set of allowed mutations at a
sequence position, wherein the nucleic acid molecule has an
acceptable phenotype. In some aspects, the method is used to evolve
a protein to a desired phenotype.
[0040] An additional aspect of the disclosure provides a method of
directed evolution, the method comprising: (a) from a first
plurality of sequences, generating a first plurality of linked
sequence-phenotype pairs (e.g., via a method for sequencing a
nucleic acid molecule described herein); (b) analyzing the first
linked sequence-phenotype pairs to design a plurality of second
sequences, wherein at least one of the second sequences has a high
probability of expressing a protein having a desired phenotype; (c)
optionally generating and analyzing a second plurality of linked
sequence-phenotype pairs according to the methods of any of the
claims; and (d) optionally iterating this cycle as many times as
necessary to isolate a protein with the desired phenotype.
[0041] An additional aspect of the disclosure provides a method of
directed evolution, the method comprising: (a) generating a library
of mutant polypeptides associated with their encoding nucleic
acids; (b) applying the library to an array, whereby there is
essentially one mutant per array position; (c) measuring the
phenotype of the mutant polypeptide at each array position; (d)
sequencing at least part of the nucleic acid at each array
position; and (e) analyzing the linked phenotype data and sequence
data, wherein the linked data informs mutations suitable for
evolving the polypeptide toward a desired phenotype.
[0042] An additional aspect of the disclosure provides an apparatus
comprising an array, wherein the array is capable of sequencing
nucleic acids and measuring a phenotype of a protein.
[0043] An additional aspect of the disclosure provides an apparatus
comprising a member that collects linked sequence-phenotype data
from an array of nucleic acid-protein pairs.
[0044] In some aspects, the array comprises at least 10.sup.4
positions. In some aspects, the array comprises at least 10.sup.5
positions. In some aspects, the array comprises at least 10.sup.6
positions. In some aspects, the array comprises at least 10.sup.7
positions. In some aspects, the array comprises at least 10.sup.8
positions. In some aspects, the array comprises one or more
sensors. In some aspects, the array is interrogated by one or more
sensors. In some aspects, the one or more sensors comprise a
chemical field-effect transistor (chemFET) sensor. In some aspects,
the sensors measure a signal associated with at least one of
fluorescence, pH change and luminescence. In some aspects, the
signal is proportional to a phenotype or relatable to a phenotype
by a calibration curve. In some aspects, the signal is a change in
temperature at a given array position.
[0045] In some aspects, the mutant proteins are associated with
their encoding nucleic acid by attachment to a microbead. In some
aspects, the mutant proteins are associated with their encoding
nucleic acid by ribosome display. In some aspects, the mutant
proteins are associated with their encoding nucleic acid by RNA
display. In some aspects, the mutant proteins are associated with
their encoding nucleic acid by DNA display.
[0046] In some aspects, the phenotype is enzyme rate. In some
aspects, the phenotype is enzyme specificity. In some aspects, the
phenotype is binding affinity. In some aspects, the phenotype is
binding specificity.
[0047] In some aspects, a method further comprises contacting
proteins to a plurality of solutions comprising substrates at a
plurality of concentrations. In some aspects, a method further
comprises contacting proteins to a plurality of solutions
comprising ligands at a plurality of concentrations. In some
aspects, a method further comprises measuring the phenotype at a
plurality of temperatures. In some aspects, the phenotype is
stability when exposed to a chemical condition or a temperature. In
some aspects, the protein is expressed using cell-free protein
synthesis.
[0048] In some aspects, the protein is expressed in an emulsion. In
some aspects, the nucleic acid is amplified in an emulsion PCR. In
some aspects, the protein is labeled at a defined stoichiometry,
wherein the label is used to determine the number of proteins at
the array position. In some aspects, the protein associates with a
known stoichiometry of probe molecule on the array. In some
aspects, the probe molecule is an antibody linked to a fluorescent
molecule, an enzyme, or an enzymatic substrate. In some aspects,
the nucleic acid is sequenced more than once. In some aspects, the
nucleic acid is sequenced a plurality of times starting from
various positions along the nucleic acid sequence. In some aspects,
the nucleic acid is amplified in an emulsion PCR, wherein a
plurality of secondary nucleic acid molecules are created
corresponding to different portions of the nucleic acid, wherein
the secondary nucleic acid molecules are sequenced.
[0049] With respect to aspects of the disclosure that have been
described as a set or genus, every individual member of the set or
genus is intended, individually, as an aspect of the disclosure,
even if, for brevity, every individual member has not been
specifically mentioned herein. When aspects of the disclosure are
described herein as being selected from a genus, it should be
understood that the selection can include mixtures of two or more
members of the genus. Similarly, with respect to aspects of the
disclosure described herein as a range, such as a range of values,
every sub-range within the range is considered an aspect of the
disclosure.
[0050] Additional aspects and advantages of the present disclosure
will become readily apparent to those skilled in this art from the
following detailed description, wherein only illustrative
embodiments of the present disclosure are shown and described. As
will be realized, the present disclosure is capable of other and
different embodiments, and its several details are capable of
modifications in various obvious respects, all without departing
from the disclosure. Accordingly, the drawings and description are
to be regarded as illustrative in nature, and not as
restrictive.
INCORPORATION BY REFERENCE
[0051] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings or figures (also "FIG."
and "FIGs." herein), of which:
[0053] FIG. 1A shows a schematic illustration of an example method
for assembling sequences of individual nucleic acid molecules;
[0054] FIG. 1B shows example data that barcode pairing can improve
assembly lengths;
[0055] FIG. 1C provides example length histograms of the contiguous
sequences ("contigs") assembled from genomic reads (minimum lengths
of about 1000 bps) from E. coli MG1655 (top panel) and Gelsemium
sempervirens (bottom panel);
[0056] FIG. 2 shows an example three-dimensional scatter plot
(inset) showing barcode fidelity in sequencing results from a
mixture of three homologous 3-kb plasmids (i.e., three target
nucleic acid molecules);
[0057] FIG. 3 is a detailed schematic of an example conversion of
sheared circular DNA into a sequencing-ready library;
[0058] FIG. 4 is a schematic diagram showing example linear
amplification of nucleic acid sequence prior to exponential PCR to
reduce amplification bias;
[0059] FIG. 5 is a schematic diagram showing an example approach
used to attach the same barcode to both ends of a target
molecule;
[0060] FIG. 6 is a schematic diagram of showing another example
approach used to attach the same barcode to both ends of a target
molecule, by creating a circularizing barcode adapter containing
two full copies of the same degenerate barcode;
[0061] FIG. 7 is a schematic diagram showing an example approach
for incorporating barcodes to full-length cDNA during
reverse-transcription;
[0062] FIG. 8A is a schematic diagram of an example method for
fragment generation based on extension of random primers;
[0063] FIG. 8B continues from FIG. 8A and completes the example
method of fragment generation based on extension of random primers;
and
[0064] FIG. 9 schematically depicts an example computer control
system described herein.
DETAILED DESCRIPTION
[0065] While various embodiments of the invention have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions may occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed.
[0066] Aspects of the disclosure described with "a" or "an" should
be understood to include "one or more" unless the context clearly
requires a narrower meaning.
[0067] The disclosure provides an improved method for obtaining
nucleic acid sequence information. In various aspects, the method
permits the quicker and more accurate assembly of intermediate and
long read lengths of target nucleic acids from short nucleic acid
sequences.
[0068] The disclosure also provides methods for obtaining nucleic
acid sequence information by reconstructing intermediate and/or
long nucleic acid sequences from the assembly of short or
intermediate nucleic acid sequences.
[0069] FIG. 1A and FIG. 1B provide an illustration of an example
embodiment of the disclosure, and shows how barcode pairing (as
described herein) improves sequence assembly of long nucleic acid
sequences. FIG. 1A shows a schematic illustration of a method for
assembling sequences of individual nucleic acid molecules. Mixed
target molecules are tagged with tripartite adapters comprising an
outer PCR priming region (black bar), an inner region containing a
sequencing primer region (burlap bars), and a central degenerate
barcode region (diagonal bars and diamond bars). PCR is carried out
generating many copies of each tagged molecule (1 in FIG. 1A). The
priming region is removed by enzymatic digestion and a single break
(on average) is made in each copy of the tagged molecule (2 in FIG.
1A). Tagged nucleic acid molecules are circularized (3a in FIG. 1A)
bringing the newly exposed end of the fragment into proximity with
the barcode. Circularized, tagged nucleic acid molecules are
linearized; a second sequencing primer/adapter (grey bar) is added;
and sequencing-ready libraries are prepared (4a in FIG. 1A).
Sequence reads begin with the barcode sequence and continue into
the unknown region. Short reads are grouped by common barcodes to
assemble the original target molecule (5a in FIG. 1A). A
barcode-pairing protocol (grey box) is used to resolve the two
distinct barcodes affixed to each original target molecule.
Circularization of unbroken copies (3b in FIG. 1A) brings the two
barcodes together. Subsequent sequencing reads contain both barcode
sequences (4b in FIG. 1A), allowing the two barcode-defined groups
to be collapsed into a single group (5b in FIG. 1A).
[0070] FIG. 1B shows that barcode pairing cam improve assembly
lengths. Reads associated with two distinct barcodes are shown
aligned to the MG1655 reference genome. Individually, each group of
reads (top) assembles into a contiguous sequence ("contig") about 6
kb in length. Barcode pairing merges the groups (bottom),
increasing and smoothing coverage across the region to allow
assembly of the full 10-kb target sequence. FIG. 1C provides length
histograms of the contigs assembled from genomic reads (minimum
length of about 1000 bp) from E. coli MG1655 (top panel) and
Gelsemium sempervirens (bottom panel). The N50 length of the
synthetic reads for E. coli MG1655 is 6.0 kb, and the longest
synthetic read (contig) in this example is 11.6 kb. The N50 length
of the synthetic reads is 4.0 kb.
[0071] FIG. 2 shows an example three-dimensional scatter plot
(inset) showing barcode fidelity in sequencing results from a
mixture of three homologous 3-kb plasmids (i.e., three target
nucleic acid molecules). The reads associated with each barcode
were searched for short sequences unique to each variant. Each
point represents a different barcode (about 8000 total) and its
position indicates the number of times sequences unique to each of
three mixed target molecules were found within that set of
barcode-grouped reads. Counting the barcodes associated with each
target molecule provides a measurement of mixture composition.
Although Target 3 was rare in the mixture, the barcodes that tagged
Target 3 had as many counts as barcodes tagging more abundant
targets.
[0072] FIG. 3 is a detailed schematic of an aspect of the
disclosure showing example conversion of sheared circular DNA into
a sequencing-ready library. Circularized DNA (black) containing
barcode and annealing sequences (grey) is fragmented (dotted line)
into molecules of about 500 bp in length. Some of the resulting
molecules will contain a barcode and others will not. Asymmetric
adapters are ligated to each end of the molecules. Limited-cycle
PCR is performed with a first primer complementary to the
asymmetric adapter and a second primer complementary to the
internal annealing sequence from the tripartite adapter. The
primers add the full sequencing adapter sequences to the PCR
product. Only molecules containing internal annealing sequences and
barcodes are exponentially amplified in the PCR.
[0073] FIG. 4 is a schematic diagram of an aspect of the disclosure
showing example linear amplification of nucleic acid sequence prior
to exponential PCR to reduce amplification bias. In some aspects,
the tripartite adapter is designed with an overhang containing an
annealing region for a linear amplification primer (grey arrow).
Each round of thermocycling in the presence of this primer copies
the original adapter ligated molecules. However, the newly
synthesized copies will not themselves be copied because they do
not have the annealing site for the linear amplification primer.
Exponential PCR can be triggered by the addition of a second primer
(black arrow).
[0074] FIG. 5 is a schematic diagram of an aspect of the disclosure
showing an example approach used to attach the same barcode to both
ends of a target molecule. An oligonucleotide is synthesized
containing a uracil base (white circle) and a degenerate barcode
region (grey region). A second oligonucleotide is synthesized to
contain a uracil base and to be complementary to a region of the
first oligonucleotide. The second oligonucleotide anneals to the
first and is extended by a DNA polymerase, copying the barcode
region and forming a double-stranded molecule. The target molecule
is circularized around the double-stranded adapter. USER enzyme
excises the uracil bases, creating nicks in each strand, and
opening the circular molecule into a linear molecule. DNA
polymerase extends the new 3' ends, copying the single-stranded
barcode regions to create a fully double-stranded molecule. An
additional adapter containing a PCR primer annealing sequence is
ligated to both ends of the linear molecule. The end result is a
linear molecule with the same barcode on both ends.
[0075] FIG. 6 is a schematic diagram of an aspect of the disclosure
showing another example approach used to attach the same barcode to
both ends of a target molecule, by creating a circularizing barcode
adapter containing two full copies of the same degenerate barcode.
An oligonucleotide (i.e., "oligo") is synthesized to contain a
nicking endonuclease site (black circle), a degenerate barcode
(grey), a self-priming hairpin, and two or more uracil bases (white
circles). The self-priming 3' end is extended with DNA polymerase,
copying the barcode sequence. The DNA is nicked at the newly
double-stranded nicking endonuclease site, creating a free 3' end.
The free 3' end is extended by a strand-displacing DNA polymerase,
which copies the barcode sequence yet again. The target molecule is
circularized around the barcode adapter by ligation. In some
aspects, USER enzyme excises two or more uracils from the original
synthetic strand, creating a single-strand gap. S1 nuclease or mung
bean nuclease degrades the single-stranded DNA, opening the circle
into a linear molecule with identical barcodes at both ends.
[0076] FIG. 7 is a schematic diagram of an aspect of the disclosure
showing an example approach for incorporating barcodes to
full-length cDNA during reverse-transcription. (1) RNA (white) is
reverse transcribed (RT) from a primer comprising an annealing
portion (grey) and a tripartite overhang portion (black) containing
a barcode. (2) Following 1st strand synthesis, the RNA is degraded
by RNase treatment and excess primers are removed. (3) A second
tripartite barcode-containing primer is added and the 2nd strand is
synthesized. (4) Excess primers are removed, and full-length cDNA
is exponentially amplified by PCR with a third primer (black
arrows) complimentary to adapters on both strands.
[0077] FIG. 8A and FIG. 8B schematically depict an alternate,
example approach to creating fragments that relies on extension of
random primers rather than breaking full-length copies. Following
adapter attachment and optional PCR, the strands are denatured and
random primers are annealed along the length of the target
molecule. The primers can be designed with a random sequence at the
3' end (e.g., N.sub.4 to N.sub.8) and optionally a defined sequence
at the 5' end that is the reverse complement of the sequence at the
ends of the target molecule (denoted by "X" in the figure) and
contains uracil bases. Extension of the random primers with a
strand-displacing polymerase creates single-stranded fragments with
one random end defined by the annealing site of the random primer
and a second end defined by the termination of extension at end of
the target fragment. Second-strand synthesis with an additional
primer with a sequence corresponding to X and containing one or
more uracil bases can create double-stranded fragments. Both
extension rounds can be performed at a relatively high temperature
to prevent further annealing of the random primers. The
double-stranded fragments can be circularized by blunt-end
ligation, or if the X-complementary overhangs were used, USER
enzyme mix (New England Biolabs) can be used to excise the
uracil-containing regions to produce sticky ends to increase
circularization efficiency.
Nucleic Acids and Nucleic Acid Libraries
[0078] A nucleic acid or nucleic acid molecule, as used herein, can
include any nucleic acid of interest. In some embodiments, nucleic
acids include, but are not limited to, DNA, RNA, peptide nucleic
acid, morpholino nucleic acid, locked nucleic acid, glycol nucleic
acid, threose nucleic acid, mixtures thereof, and hybrids thereof.
In some aspects, a nucleic acid is a "primer" capable of acting as
a point of initiation of synthesis along a complementary strand of
nucleic acid when conditions are suitable for synthesis of a primer
extension product.
[0079] In some aspects, the nucleic acid serves as a template for
synthesis of a complementary nucleic acid, e.g., by
base-complementary incorporation of nucleotide units. For example,
in some aspects, a nucleic acid comprises naturally occurring DNA
(including genomic DNA), RNA (including mRNA), and/or comprises a
synthetic molecule including, but not limited to, complementary DNA
(cDNA) and recombinant molecules generated in any manner. In some
aspects, the nucleic acid is generated from chemical synthesis,
reverse transcription, DNA replication or a combination of these
generating methods. In some aspects, the linkage between the
subunits is provided by phosphates, phosphonates, phosphoramidates,
phosphorothioates, or the like, or by nonphosphate groups, such as,
but not limited to, peptide-type linkages utilized in peptide
nucleic acids (PNAs). In some aspects, the linking groups are
chiral or achiral. In some aspects, the polynucleotides have any
three-dimensional structure, encompassing single-stranded,
double-stranded, and triple helical molecules that are, e.g., DNA,
RNA, or hybrid DNA/RNA molecules, and double-stranded with
single-stranded regions (for example, stem- and
loop-structures).
[0080] In some aspects, nucleic acids are obtained from any source.
In various aspects, nucleic acid molecules are obtained from a
single organism or from populations of nucleic acid molecules
obtained from natural sources that include one or more organisms.
Sources of nucleic acid molecules include, but are not limited to,
organelles, cells, tissues, organs, and organisms. In some aspects,
when cells are used as sources of nucleic acid molecules, the cells
are derived from any prokaryotic or eukaryotic source. Such cells
include, but are not limited to, bacterial cells, fungal cells,
plant cells (including vegetable cells), protozoan cells, and
animal cells. Such animal cells include, but are not limited to,
insect cells, nematode cells, avian cells, fish cells, amphibian
cells, reptilian cells, and mammalian cells. In some aspects, the
mammalian cells include human cells.
[0081] Nucleic acids can be obtained using any suitable method,
including those described by Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). In
some aspects, nucleic acids are obtained as described in U.S.
Patent Application Publication No. US2002/0190663. Nucleic acids
obtained from biological samples typically are fragmented to
produce suitable fragments for analysis.
[0082] In some aspects, a nucleic acid of interest or "target
nucleic acid" or "target nucleotide sequence" to be sequenced is
fragmented or sheared to a desired length. The terms "fragmenting,"
"shearing," or "breaking" are used interchangeably in various
aspects herein to mean cutting or cleaving the nucleic acid into at
least two or more smaller pieces or fragments. In various aspects,
a nucleic acid is shortened, or broken into fragments of shorter
lengths, in the preparation of a high quality sequencing library or
"target library," which is important in next-generation sequencing
(NGS). In various embodiments, a "target library" or "target
nucleic acid library" is created. The target library comprises
fragments of a target nucleic acid of interest. The term "target
nucleic acid" or "target nucleotide" or "target nucleotide
sequence" is used herein interchangeably to refer to the nucleic
acid or nucleotide to be sequenced.
[0083] In various aspects, a nucleic acid is fragmented or
shortened by physical, chemical, or enzymatic shearing. In various
aspects, physical fragmentation is carried out by acoustic
shearing, sonication, or hydrodynamic shear. In many aspects,
acoustic shearing and sonication are popular physical methods used
to shear DNA. In some aspects, the Covaris.RTM. instrument
(Covaris, Woburn, Mass.) is an acoustic device used for breaking
DNA into fragments of about 100 bp to about 5000 bp. In other
aspects, the Bioruptor.RTM. (Denville, N.J.) is a sonication device
utilized for shearing chromatin, DNA and disrupting tissues. Small
volumes of DNA are sheared to about 150 to about 1 kb in length.
Hydroshear (Digilab, Marlborough, Mass.) utilizes hydrodynamic
forces to shear DNA. In some aspects, DNA is sheared by nebulizers
(Life Tech, Grand Island, N.Y.), which atomize liquid using
compressed air, and results in shearing DNA into fragments of about
100 bp to about 3000 bp in seconds. In various aspects, enzymatic
fragmentation or shearing is carried out by fragmentase (NEB,
Ipswich, Mass.), KAPA Frag Enzyme (KAPA, Wilmington, Mass.), DNase
I, non-specific nuclease, transposase, another restriction
endonuclease, or Nextera tagmentation technology (Illumina, San
Diego, Calif.). In various aspects, chemical fragmentation is
carried out. Chemical fragmentation includes, but is not limited
to, exposure to heat and divalent metal cations. Chemical shear is
typically reserved for the breakup of long RNA fragments, and is
typically performed through the heat digestion of RNA with a
divalent metal cation (magnesium or zinc). In some aspects, the
length of the RNA (about 115 nucleotides to about 350 nucleotides)
is adjusted by increasing or decreasing the time of incubation. In
some aspects, a nucleic acid molecule is shortened with an
exonuclease.
[0084] In various aspects, the size of the nucleic acid fragment is
a key factor for library construction and sequencing. In various
aspects, a sequencing platform and read length is chosen to be
compatible with fragment size. In some aspects, size selection of
nucleic acids is performed to remove very short fragments or very
long fragments.
[0085] In various aspects, fragmentation is carried out in various
stages of the method disclosed herein. For example, in some
aspects, there are three fragmentation rounds. For example, in some
aspects, if genomic DNA is used as a starting material (rather than
mRNA or a PCR product), genomic DNA is fragmented in a first
fragmentation into pieces of about 8 kb to about 10 kb. Those
fragments of about 8 kb to about 10 kb are tagged and amplified.
The amplified copies, in various aspects, are further fragmented in
a second fragmentation, ideally breaking them one time somewhere
along their length into fragments of various lengths. These
fragments of various lengths are circularized, and the circles are
fragmented again in a third fragmentation to fragments of about 300
bases to about 800 bases.
[0086] In various aspects, therefore, fragment size is about 0.1
kilobase (kb), about 0.15 kb, about 0.2 kb, about 0.25 kb, about
0.3 kb, about 0.35 kb, about 0.4 kb, about 0.45 kb, about 0.5 kb,
about 0.55 kb, about 0.6 kb, about 0.65 kb, about 0.7 kb, about
0.75 kb, about 0.8 kb, about 0.85 kb, about 0.9 kb, about 0.95 kb,
about 1.0 kb, about 1.5 kb, about 2.0 kb, about 2.5 kb, about 3.0
kb, about 3.5 kb, about 4.0 kb, about 4.5 kb, about 5.0 kb, about
5.5 kb, about 6.0 kb, about 6.5 kb, about 7.0 kb, about 7.5 kb,
about 8.0 kb, about 8.5 kb, about 9.0 kb, about 9.5 kb, about 10
kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb, about 15
kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, about 20
kb, about 30 kb, about 40 kb, about 50 kb, about 60 kb, about 70
kb, about 80 kb, about 90 kb, about 100 kb, about 1000 kb, or
longer.
[0087] In various aspects, size selection is carried out. In some
aspects, size-selection is used, after shearing genomic DNA into
large fragments, to separate desirable nucleic acid fragments of a
size of about 8 kb to about 10 kb from smaller fragments, which
would preferentially amplify during PCR and ultimately yield
synthetic reads of limited usefulness. In some aspects, size
selection is used after fragmentation of PCR products to enrich the
library for fragments of a particular size to compensate for
diminished circularization efficiency of fragments depending on
size. In some aspects, circularization efficiency is reduced if
fragment length is too long.
[0088] In some aspects, size selection is carried out using
length-dependent binding to solid phase reversible immobilization
(SPRI.RTM., Beckman Coulter) beads. In other aspects, size
selection is carried out using agarose or polyacrylamide
electrophoresis gel purification and isolation. Size selection via
gel electrophoresis purification and isolation may be performed
manually or with an automated system such as BluePippen (Sage
Science, Beverly, Mass.) or E-gels (Thermo Fisher Scientific)
[0089] The term "long nucleotide sequence," "long nucleic acid
sequence," or "long read" as used herein refers to any nucleic acid
sequence equal to or greater than 20,000 bases (or 20,000
nucleotides, or 20 kilobases, or 20 kb). In some aspects, the long
nucleotide sequence is between approximately 20000 bases to
approximately 500,000 bases. In some aspects, the long nucleotide
sequence is between approximately 25000 bases to approximately
100,000 bases. In some aspects, the long nucleotide sequence is
about 20000 bases, about 25000 bases, about 30000 bases, about
35000 bases, about 40000 bases, about 45000 bases, about 50000
bases, about 55000 bases, about 60000 bases, about 65000 bases,
about 70000 bases, about 75000 bases, about 80000 bases, about
85000 bases, about 90000 bases, about 95000 bases, about 100,000
bases, about 150,000 bases, about 200,000 bases, about 250,000
bases, about 300,000 bases, about 350,000 bases, about 400,000
bases, about 450,000 bases, or about 500,000 bases.
[0090] The term "intermediate nucleotide sequence," "intermediate
nucleic acid sequence," or "intermediate read" as used herein
refers to any nucleic acid sequence greater than 1000 bases and
less than 20,000 bases. In some aspects, the intermediate
nucleotide sequence is between approximately 1500 bases and
approximately 15000 bases. In some aspects, the intermediate
nucleotide sequence is between approximately 2000 bases to
approximately 12000 bases. In some aspects, the intermediate
nucleotide sequence is between approximately 3000 bases to
approximately 11000 bases. In some aspects, the intermediate
nucleotide sequence is between approximately 4000 bases to
approximately 10000 bases. In some aspects, the intermediate
nucleotide sequence is about 1050 bases, about 1100 bases, about
1150 bases, about 1200 bases, about 1250 bases, about 1300 bases,
about 1350 bases, about 1400 bases, about 1450 bases, about 1500
bases, about 1550 bases, about 1600 bases, about 1650 bases, about
1700 bases, about 1750 bases, about 1800 bases, about 1850 bases,
about 1900 bases, about 1950 bases, about 2000 bases, about 2100
bases, about 2200 bases, about 2300 bases, about 2400 bases, about
2500 bases, about 3000 bases, about 3500 bases, about 4000 bases,
about 4500 bases, about 5000 bases, about 5500 bases, about 6000
bases, about 6500 bases, about 7000 bases, about 7500 bases, about
8000 bases, about 8500 bases, about 9000 bases, about 9500 bases,
about 10000 bases, about 11000 bases, about 12000 bases, about
13000 bases, about 14000 bases, about 15000 bases, about 16000
bases, about 17000 bases, about 18000 bases, about 19000 bases, or
less than about 20000 bases.
[0091] The term "short nucleotide sequence," "short nucleic acid
sequence," or "short read" as used herein refers to any nucleic
acid sequence less than or equal to 1000 bases or 1000 nucleotides.
In some aspects, the short nucleotide sequence is between
approximately 25 bases to approximately 1000 bases. In some
aspects, the short nucleotide sequence is between approximately 50
bases to approximately 750 bases. In some aspects, the short
nucleotide sequence is between approximately 75 bases to
approximately 500 bases. In some aspects, the short nucleotide
sequence is about 25 bases, about 50 bases, about 75 bases, about
100 bases, about 125 bases, about 150 bases, about 175 bases, about
200 bases, about 250 bases, about 275 bases, about 300 bases, about
325 bases, about 350 bases, about 375 bases, about 400 bases, about
425 bases, about 450 bases, about 475 bases, about 500 bases, about
525 bases, about 550 bases, about 575 bases, about 600 bases, about
675 bases, about 700 bases, about 725 bases, about 750 bases, about
775 bases, about 800 bases, about 825 bases, about 850 bases, about
875 bases, about 900 bases, about 925 bases, about 950 bases, about
975 bases, or about 1000 bases.
Adapters and Adapter Attachment
[0092] An "adapter" as used herein is a relatively short, nucleic
acid molecule which is attached to a nucleic acid molecule in
various aspects of the disclosure. In some aspects, an adapter
comprises a variety of sequence elements including, but not limited
to, an amplification primer annealing sequence or complement
thereof, a sequencing primer annealing sequence or complements
thereof, a barcode sequence, a common sequence shared among
multiple different adapters or subsets of different adapters, a
restriction enzyme recognition sites, an overhang complementary to
a target polynucleotide overhang, a probe binding site (e.g., for
attachment to a sequencing platform), a random or near-random
sequence (e.g., a nucleotide selected at random from a set of two
or more different nucleotides at one or more positions, with each
of the different nucleotides selected at one or more positions
represented in a pool of adapters comprising the random sequence),
and combinations thereof. In some aspects, two or more sequence
elements are non-adjacent to one another (e.g., separated by one or
more nucleotides), adjacent to one another, partially overlapping,
or completely overlapping. In some aspects, adapters contain
overhangs designed to be complementary to a corresponding overhang
on the molecule to which ligation is desired. In some aspects, a
complementary overhang is one or more nucleotides in length
including, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, or more nucleotides in length. In some aspects, a
complementary overhang comprises a fixed or a random sequence.
[0093] In some aspects, the adapter is a "tripartite adapter"
comprising a polymerase chain reaction (PCR) primer region, a
sequencing primer region, and a barcode region. In some further
aspects, the tripartite adapter comprises an outer PCR primer
region (or amplification primer region or sequence), an inner
sequencing primer region (or sequence), and a central barcode
region (or sequence). Barcodes are important in solving the problem
of information loss resulting from the shearing of a target nucleic
acid into sequencing-compatible fragments. In some aspects, each
barcode is specific to the individual intermediate-length nucleic
acid molecule from which a given short sequenced nucleic acid
molecule is derived and is used to identify the source of the short
nucleic acid. In various aspects, therefore, a given barcode is
exclusively associated with a single target molecule. Thus, the
term "barcode fidelity" as used herein refers to a particular
barcode being exclusively associated with a single target molecule.
With perfect barcode fidelity, every read tagged with that barcode
is derived from that single target molecule and contains nucleotide
sequence from that single target molecule alone. Thus, when being
assembled (e.g., in a computational pipeline), reads sharing a
barcode sequence are distinguished from the background of reads
without that particular barcode, and are grouped together and
assembled to recreate the sequence of the original longer molecule.
A "computational pipeline" or "processing pipeline" is a system for
processing sequencing data and assembling the short nucleic acid
sequence data into synthetic long nucleic acids.
[0094] In some aspects, short defined sequences are designed to
follow and/or precede the barcode sequence in the sequencing reads
to positively distinguish true barcode sequences from spurious
sequences. In some aspects, these constant sequences are selected
to promote incorporation of biotinylated deoxyribonucleotides
(e.g., biotin-dCTP) into the fragmented molecules during
end-repair.
[0095] In some aspects, an amplification primer annealing sequence
also serves as a sequencing primer annealing sequence. In some
aspects, sequence elements are located at or near the ligating end,
at or near the non-ligating end, or in the interior of the adapter.
In some aspects, when an adapter oligonucleotide is capable of
forming secondary structure, such as a hairpin, sequence elements
are located partially or completely outside the secondary
structure, partially or completely inside the secondary structure,
or in between sequences participating in the secondary structure.
For example, in some aspects, when an adapter oligonucleotide
comprises a hairpin structure, sequence elements are located
partially or completely inside or outside the hybridizable
sequences (the "stem"), including in the sequence between the
hybridizable sequences (the "loop").
[0096] In some aspects, the first adapter oligonucleotides in a
plurality of first adapter oligonucleotides having different
barcode sequences comprise a sequence element common among all
first adapter oligonucleotides in the plurality. In some aspects,
all second adapter oligonucleotides comprise a sequence element
common among all second adapter oligonucleotides that is different
from the common sequence element shared by the first adapter
oligonucleotides. In some aspects, a difference in sequence
elements is any such difference, wherein at least a portion of
different adapters do not completely align, for example, due to
changes in sequence length, deletion, or insertion of one or more
nucleotides, or a change in the nucleotide composition at one or
more nucleotide positions (such as a base change or base
modification).
[0097] In some aspects, partial sequencing primer sequences (e.g.,
like those available from Illumina) are included adjacent to the
random barcode sequence in the barcode adapter. In some aspects,
the partial sequence anneals in downstream PCR to a longer
oligonucleotide that adds a full sequencing primer sequence (e.g.,
like those available from Illumina). Alternatively, in some
aspects, other sequences are used with a corresponding custom
sequence primer in place of a standard sequencing primer
mixture.
[0098] In some aspects, the adapter comprises sequencing primer
sequence proximal to the barcode. This positioning of the
sequencing primer and the barcode provides two main benefits.
First, because the sequencing read (e.g., Illumina) begins with the
sequence directly downstream of the sequencing primer sequence, the
barcode sequence is always located at the beginning of one of the
two paired-end sequencing reads (e.g., Illumina). After the barcode
sequence, the read continues directly into an unknown region
derived from the middle of the target molecule. This positioning of
the barcode and sequencing primer ensures that the random barcode
is easily identifiable, and avoids wasting sequencing capacity by
repeatedly sequencing the region on the upstream side of the
barcode (which is always derived from the end of the original
target molecule). Second, the presence of a primer sequence (e.g.,
Illumina) adjacent to the barcode sequence provides a simple way to
distinguish nucleic acid fragments containing barcodes from
fragments that do not contain barcodes. In some aspects, these
latter fragments arise when a copy of the amplified target molecule
is broken more than once, creating two end fragments with barcode
sequences and one or more middle fragments without barcodes. In
these instances, sequencing barcode-free fragments wastes
sequencing capacity, because they contain no barcode sequence to
link them to a parent nucleic acid molecule. In some aspects, only
end fragments containing barcode sequences contain the primer
sequences (e.g., Illumina) that are used to selectively amplify
these sequences by PCR.
[0099] In some aspects, an asymmetric adapter is ligated to both
ends of a nucleic acid fragment (see FIG. 3). In some aspects, this
ligation of an asymmetric adapter takes place following
fragmentation, circularization, and shearing. In some aspects, this
asymmetric adapter comprises two oligonucleotides, one of which is
longer than the other. In some aspects, the shorter oligonucleotide
is complementary to the longer oligonucleotide and, upon annealing,
creates a ligation-competent adapter with a 3' dT-tail suitable for
specific ligation to the A-tailed fragment. In some aspects, the
adapter sequence is complementary to a PCR primer that adds a
second sequencing primer sequence (e.g., Illumina) by
overlap-extension PCR, but only the longer of the two
oligonucleotides is long enough to productively anneal to this
primer during PCR. As a result, following ligation of an asymmetric
adapter to both ends of a fragment, each of the two strands of the
fragment has an annealing-competent sequence at only one end. The
second PCR primer in the reaction anneals to the partial sequence
(e.g., Illumina) contained within the fragment adjacent to the
barcode. As a result, the only exponentially amplified PCR product
is the desired nucleic acid fragment, which begins with one
sequence (e.g., Illumina), followed by the barcode sequence and
unknown sequence from the center of the target molecule, and ends
with the second sequence (e.g., Illumina). Fragments of about 500
bp are converted into a sequenceable library by adding any
requisite binding sequences (e.g., Illumina flowcell binding
sequences) to the ends of the fragments.
[0100] In some aspects, library preparation is similar to library
preparation carried out with commercial (e.g., Illumina) reagents
(e.g., which is done with forked or Y-shaped adapters that ensure
that the PCR-amplified products all have adapter 1 on one end and
adapter 2 on the other end); however, in the method of the
disclosure one of the forks of the Y-shaped adapter is omitted
because the fragments of interest already contain an annealing site
for one of the two sequencing primers. Therefore, in some aspects,
one primer anneals to the remaining fork, and the other primer
anneals to a site in the interior of the fragment. In some aspects,
therefore, (e.g., Illumina) sequences are used to ensure
compatibility with standard sequencing reagents (e.g., Illumina)
used in the sequencing methods. In some aspects, therefore,
sequencing is carried out using a number or variety of sets of
sequences (e.g., TruSeq kit, Small RNA kit, and the like, any of
which are useful in various aspects described herein.
[0101] In some aspects, an adapter comprises a region that is
identical among all members of the adapter population and a
degenerate barcode region that is unique to each member of the
population. In general, a barcode comprises a nucleic acid sequence
that when observed together with a polynucleotide serves as an
identifier of the sample or molecule from which the polynucleotide
was derived. As used herein, the term "barcode" refers to a nucleic
acid sequence that allows some feature of a polynucleotide with
which the barcode is associated to be identified. In some aspects,
the feature of the polynucleotide to be identified is the sample or
molecule from which the polynucleotide is derived. In some aspects,
barcodes are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, or more nucleotides in length. In some aspects, barcodes are
shorter than 10, 9, 8, 7, 6, 5, or 4 nucleotides in length. In some
aspects, barcodes associated with some polynucleotides are of
different lengths than barcodes associated with other
polynucleotides. In general, barcodes are of sufficient length and
comprise sequences that are sufficiently different to allow the
identification of samples based on barcodes with which they are
associated. In some aspects, a barcode, and the sample source with
which it is associated, is identified accurately after the
mutation, insertion, or deletion of one or more nucleotides in the
barcode sequence, such as the mutation, insertion, or deletion of
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. In some
aspects, each barcode in a plurality of barcodes differ from every
other barcode in the plurality by at least two nucleotide
positions, for example, by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more positions. In some aspects, both a first adapter and a second
adapter comprise at least one of a plurality of barcode sequences.
In some aspects, barcodes for second adapter oligonucleotides are
selected independently from barcodes for first adapter
oligonucleotides.
[0102] In some aspects, the tripartite adapter further comprises an
index sequence to facilitate multiplexing of more than one sample
for simultaneous preparation and sequencing. As opposed to the
barcode region, the index region is not degenerate but defined, and
a set of distinct oligonucleotides are synthesized such that each
contain a different index sequence. Index sequences are long enough
to uniquely distinguish them from one another, or long enough to
uniquely distinguish them even if one or more errors are made
during sequencing. In some aspects, typical lengths for the index
sequence are 2-8 bases. In some aspects, the index sequence is
located to one side or the other of the degenerate barcode region,
between the two priming regions, and is read along with the barcode
in a single or a paired-end read. In other aspects, the index
sequence is 5' of the sequencing primer region in the synthesized
oligonucleotide and 3' of an additional sequence that anneals to
oligonucleotides attached to the sequencing flowcell (or that
anneals to a primer that adds such a sequence during PCR). In this
aspect, the adapter is designed to mimic the structure of a
sequencing-ready molecule, and the index is read by a separate
index read on a sequencing machine (e.g., Illumina).
[0103] In some aspects, as an alternative to downstream linkage of
two distinct barcode sequences ligated to the two ends of the
target molecule, both ends of the target molecule are tagged with
the same barcode sequence.
[0104] In some aspects, a single circularization barcode adapter is
ligated to the target molecule in lieu of two end adapters. In some
aspects, the two ends of this adapter ligate to the two ends of the
same target molecule to form a circular molecule.
[0105] In some aspects, the adapter contains a single barcode
sequence, which is flanked in the 5' direction on each strand by
uracil bases (see FIG. 5). In some aspects, after circularization,
the USER.TM. enzyme mix (Uracil-Specific Excision Reagent) Enzyme
(NEB) excises uracils and breaks the phosphate backbone. The term
"USER enzyme" as used herein refers to USER.TM. (NEB), which is a
mixture of Uracil DNA glycosylase (UDG) and the DNA
glycosylase-lyase Endonuclease VIII. UDG catalyzes the excision of
a uracil base, forming an abasic (apyrimidinic) site while leaving
the phosphodiester backbone intact. The lyase activity of
Endonuclease VIII breaks the phosphodiester backbone at the 3' and
5' sides of the abasic site so that base-free deoxyribose is
released. Each strand is thereby broken 5' of the barcode sequence,
opening the circular molecule into a linear molecule with 5'
single-stranded overhangs at each end that contain the same barcode
sequence. In some aspects, extension of the 3' ends by, e.g.,
Klenow exo-DNA polymerase copies the barcode sequence at each end,
creating a fully double-stranded DNA molecule with the same barcode
sequence at both ends. Klenow exo-DNA polymerase extension also
leaves single dA-tails useful for ligating additional adapters
containing sequences that serve as PCR primer annealing sites for
subsequent PCR amplification.
[0106] In some aspects, a single circularizing adapter that
contains two double-stranded copies of the same barcode sequence is
ligated to the target molecule (see FIG. 6). In some aspects, such
an adapter is prepared by synthesizing an oligonucleotide
containing a degenerate barcode region and a region that forms a
self-priming hairpin, extending the self-primed 3' end with DNA
polymerase, nicking the newly double-stranded molecule with a
nicking endonuclease at a site near the 5' end of the original
oligonucleotide, and extending the exposed 3' end with a
strand-displacing DNA polymerase. In some aspects, after
circularizing ligation to a target molecule, the adapter is cut at
a specific site between the two copies of the barcode by a
restriction enzyme or a combination of USER enzyme and a nuclease
that specifically digests single-stranded DNA, such as S1 nuclease
or mung bean nuclease.
[0107] In some aspects, an adapter comprising two copies of the
same barcode is used. After circularization around the adapter,
USER enzyme or another nuclease breaks the adapter between the
barcode copies, yielding a linear molecule with the same barcode at
both ends. A schematic of this approach is set out in FIG. 6. In
some aspects, simultaneous fragmentation and adapter addition are
carried out. In particular aspects, this simultaneous process is
carried out by the use of transposases, which are discussed herein
below in more detail.
[0108] In some aspects, adapter oligonucleotides are any suitable
length. In some aspects, the length of the adapter is at least
sufficient to accommodate the one or more sequence elements of
which the adapter comprises. In some aspects, adapters are about,
less than about, or more than about 10, about 15, about 20, about
25, about 30, about 35, about 40, about 45, about 50, about 55,
about 60, about 65, about 70, about 75, about 80, about 90, about
100, about 120, about 140, about 160, about 180, about 200, about
300, about 400, about 500, about 600, about 700, about 800, about
900, or more nucleotides in length. In more particular aspects,
adapters are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, or 60 nucleotides in length.
[0109] Adapter attachment can be carried out in any suitable
manner. In some aspects, an adapter is attached to each end of each
member of the target library. In some aspects, an adapter is
attached to only one end of each member of the target library. In
some aspects, an adapter is attached to the nucleic acid following
end-repair and any of dT-tailing, dA-tailing, dG-tailing, or
dC-tailing. Tailing can be performed by Klenow exo' polymerase or
Taq polymerase to add a single tailing nucleotide, or by terminal
transferase to add multiple tailing nucleotides. In some aspects,
the adapter is attached by ligation. The term "ligation" as used
herein, with respect to two polynucleotides, refers to the covalent
attachment or joining of two separate polynucleotides to produce a
single larger polynucleotide with a contiguous backbone. Methods
for joining two polynucleotides include without limitation,
enzymatic and non-enzymatic (e.g., chemical) methods. Examples of
ligation reactions that are non-enzymatic include the non-enzymatic
ligation techniques described in U.S. Pat. Nos. 5,780,613 and
5,476,930, which are herein incorporated by reference. In some
embodiments, an adapter oligonucleotide is joined to a target
polynucleotide by a ligase, for example a DNA ligase or RNA ligase.
Multiple ligases, each having characterized reaction conditions
include, without limitation NAD-dependent ligases including tRNA
ligase, Taq DNA ligase, Thermus filiformis DNA ligase, Escherichia
coli DNA ligase, Tth DNA ligase, Thermus scotoductus DNA ligase (I
and II), thermostable ligase, Ampligase thermostable DNA ligase,
VanC-type ligase, 9.degree. N DNA Ligase, Tsp DNA ligase, and novel
ligases discovered by bioprospecting; ATP-dependent ligases
including T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA
ligase, Pfu DNA ligase, DNA ligase 1, DNA ligase III, DNA ligase
IV, and genetically engineered variants thereof.
[0110] In some aspects, an adapter is ligated to each end of each
double-stranded fragment of the target library. In particular
aspects, a first tripartite adapter comprising an outer PCR primer
region, an inner sequencing primer region, and a central barcode
region is attached to each end of a short, linear nucleic acid
sequence of the fragment library to form multiple barcode-tagged
fragments or sequences, wherein the first adapter attached at the
one end comprises a different barcode than the first adapter
attached at the other end.
[0111] In some aspects, the addition of adapters occurs in a mixed
solution and does not require physical separation of the nucleic
acid in order to add the adapter. Thus, in various aspects,
adapters are added to up to a million or more nucleic acids.
[0112] In some aspects, ligation is between polynucleotides having
hybridizable sequences, such as complementary overhangs. The term
"complementary" as used herein refers to a nucleic acid sequence of
bases that can form a double-stranded nucleic acid structure by
matching base pairs. In some aspects, ligation is between
polynucleotides comprising two blunt ends. In some aspects, a 5'
phosphate is utilized in a ligation reaction. In some aspects, a 5'
phosphate is provided by the target polynucleotide, the adapter
oligonucleotide, or both. In some aspects, 5' phosphates are added
to or removed from polynucleotides to be joined, as needed. Methods
for the addition or removal of 5' phosphates include without
limitation enzymatic and chemical processes. Enzymes useful in the
addition and/or removal of 5' phosphates include, but are not
limited to, kinases, phosphatases, and polymerases.
Nucleic Acid Amplification and Amplification Bias
[0113] In some embodiments, adapter-tagged target molecules are
amplified using any suitable amplification method. Amplification as
used herein refers to production of additional copies of a nucleic
acid sequence, and can be carried out using PCR or any other
suitable amplification technology (e.g., Dieffenbach and Dveksler,
PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,
Plainview, N.Y. [1995]). Examples of suitable nucleic acid
amplification methods include, but are not limited to, PCR,
quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplex
fluorescent PCR (MF-PCR), real time PCR (RTPCR), single cell PCR,
restriction fragment length polymorphism PCR (PCR-RFLP),
PCK-RFLPIRT-PCR-IRFLP, hot start PCR, nested PCR, in situ polony
PCR, in situ rolling circle amplification (RCA), bridge PCR,
picotiter PCR, and emulsion PCR. Other suitable amplification
methods include, but are not limited to, ligase chain reaction
(LCR), transcription amplification, self-sustained sequence
replication, selective amplification of target nucleic acids,
consensus sequence primed polymerase chain reaction (CP-PCR),
arbitrarily primed polymerase chain reaction (AP-PCR), degenerate
oligonucleotide-primed PCR (DOP-PCR), and nucleic acid-based
sequence amplification (NAB SA).
[0114] In some aspects, PfuCx Turbo DNA polymerase (Agilent
Technologies, La Jolla, Calif.) or KAPA HiFi Uracil+DNA Polymerase
(Kapa Biosystems, Inc., Wilmington, Mass.) is used for PCR. These
polymerase enzymes are compatible with uracil-containing primers,
yet feature a proofreading activity that reduces the error rate
relative to Taq polymerases. In some aspects, polymerase mixtures
optimized for "long-range" PCR are used. These polymerase mixtures
usually contain a mixture of Taq polymerase with a proof-reading
polymerase. Examples include LongAmp Taq (NEB) and MasterAmp
Extra-long (Epicentre). In some aspects, a single primer is used
for PCR. Using a single primer has been shown to discourage the
accumulation of primer dimers during PCR. In some other aspects,
two or more primers are used for PCR.
[0115] In some aspects, PCR bias or "amplification bias" can be a
significant challenge when amplifying complex, heterogeneous
libraries that result from shearing genomic DNA. In some aspects,
each barcode-tagged sequence in the library is amplified to a
similar extent. In some aspects, if a subset of the target
molecules dominate the PCR, fragments derived from those molecules
are sequenced disproportionately frequently, and the yield of the
sequencing reaction suffers. In some aspects, while some level of
amplification bias is unavoidable, steps are taken to minimize
impact of amplification bias. In some aspects, bias is minimized by
supplementing the PCR reaction with betaine, DMSO, or other known
additive(s), or combinations thereof, to reduce the sequence
dependence of amplification efficiency, promoting a more even
distribution of amplified products.
[0116] In some aspects, PCR suppression effects are minimized. In
some aspects, an identical sequence is ligated at both ends of a
nucleic acid. In some aspects, upon denaturation during PCR,
complementary ends anneal to form a hairpin, potentially reducing
the efficiency of PCR. In some aspects, ligating the same adapter
to both ends of the target molecule results in identical PCR
primer-annealing, and primer-annealing sequences (e.g., Illumina
primer-annealing sequences) contribute to PCR suppression hairpins,
particularly when the two random barcode sequences in the adapters
happen to be partially complementary. To minimize this effect,
distinct PCR primer-annealing sequences and/or distinct
primer-annealing sequences (e.g., Illumina) are included in the
adapters that are attached to the two ends of the target molecule.
In various aspects, steps are taken to avoid having identical
adapters on both ends of the DNA, because when the DNA becomes
single stranded the ends can anneal to form a "panhandle" structure
that blocks PCR primer annealing. In some aspects, this addition of
primer annealing sequences is accomplished by adding a mixture of
different adapters into the ligation mixture (in which case 1/n of
the ligation products will have the same adapter on both ends,
where n is the number of distinct adapters in the mixture). In
other aspects, PCR suppression is promoted by the use of longer
adapters in order to suppress amplification of shorter fragments in
favor of longer fragments.
[0117] In some aspects, a "forked" or "Y" adapter comprising two
oligonucleotides that are only partially complementary is used. In
some aspects, such oligonucleotides anneal to form an adapter that
is double stranded and ligation competent at one end, but forks
into two non-complementary single strands at the other end. This
type of adapter is often used in standard sequencing methods (e.g.,
Illumina) and may be used in some aspects of the disclosure. The
benefit is that subsequent PCR with primers complementary to the
two strands yields products with one of the two fork sequences at
one end and the other fork sequence at the other end, which is
otherwise not possible at 100% efficiency when ligating adapters to
a library of unknown sequences. Standard sequencing protocols
(e.g., Illumina) use a mixture of sequencing primers that contains
primers compatible with different library preparation kits. Two
primer mixtures are used: a "universal" primer mix that produces
the first read, and an "index" primer mix that produces the second
or paired-end read. Therefore, by ligating two distinct universal
primer-annealing sequences or two distinct index primer-annealing
sequences to the target, PCR suppression hairpins can be avoided
while preserving the ability of fragments derived from each end to
be sequenced with the same standard (e.g., Illumina) primer
mixture.
[0118] In some additional aspects, amplification bias is reduced by
a linear amplification stage prior to exponential amplification
(see FIG. 4). During the linear PCR amplification phase, only the
initially present (original) molecules, and not the newly
synthesized copies, are copied by PCR. In some aspects, the copying
of only the original nucleic acid molecules is accomplished by
ligating barcode-containing adapters with 3' overhangs to the ends
of the target molecule, such that only one of the two strands at
each end of the ligated target molecule is capable of annealing to
a PCR primer at a set annealing temperature. In some aspects,
exponential amplification is triggered by a change in the annealing
temperature or the addition of a nested primer.
[0119] In some aspects, amplification bias is minimized by
replacing PCR with rolling-circle amplification (RCA) or
hyperbranching rolling-circle amplification (HRCA). HRCA has been
used in whole-genome amplification techniques and has been shown to
amplify mixed populations with less bias than PCR. In some aspects,
a circularization adapter is ligated to the target, such that the
two ends of the adapter ligate to the ends of the same target
molecule to form a circular molecule. In some aspects, the adapter
contains a single barcode sequence, which is flanked in the 5'
direction on each strand by nicking endonuclease recognition
sequences. In some aspects, after circularization, HRCA amplifies
the molecule in an exponential manner. In some aspects, the
resulting double-stranded DNA concatamers are broken, such as, for
example, by mechanical shearing or dsDNA fragmentase. In some
aspects, the nucleic acids are then treated with a nicking
endonuclease, which introduces single-strand breaks on each side of
the barcode. In some aspects, each strand of the barcoded section
becomes a 5' overhang at the end of the resulting fragments, and
Klenow or another polymerase is used to fill in these ends, copying
the barcode to create a blunt end ready for circularization.
[0120] In some aspects, two loop adapters are ligated to the ends
of the target to create a circular "dumbbell" structure that is
amplified by HRCA. The resulting concatamers are sheared and
digested by a nicking endonuclease.
[0121] In some aspects, in place of mechanical or enzymatic
fragmentation, random fragments are generated during amplification
by PCR or rolling-circle amplification with random (degenerate) or
partially random oligonucleotide primers (e.g., see FIG. 8A and
FIG. 8B).
[0122] In some aspects, interior regions of the amplified target
molecule are exposed prior to circularization by fragmentation
using a double-stranded DNA fragmentase enzyme mixture (NEB). This
enzyme mixture is a mixture of two enzymes that creates random
breaks in double-stranded DNA. In some aspects, KAPA Frag Enzyme is
used for fragmentation. Unlike exonucleases, fragmentation enzymes
preserve both ends of the DNA molecule, both of which give rise to
productive circular molecules. Unlike mechanical shearing,
fragmentation enzymes introduce breaks along the length of the DNA
molecule independent of the distance from an end of the molecule or
independent of the size of the molecule. Additionally, in some
aspects, the number of breaks per kilobase is adjusted for
different target molecule lengths by diluting the enzyme mixture or
adjusting the reaction time. Typically, reaction time takes about
15 minutes, but is adjusted accordingly, in various aspects,
depending on the amount of DNA, the length of the DNA (e.g., the
goal is one break per molecule, regardless of its length), and the
concentration of the enzyme.
[0123] In some aspects, adapter-tagged target molecules are
amplified by PCR using a single, uracil-containing oligonucleotide
primer that is complementary to a constant region of the adapter
lying outside of the barcode sequence, such that the barcode is
copied by the extension of the primer. In some aspects,
amplification creates many copies of each target molecule such that
each copy of the same target molecule is attached to the same
barcode sequence unique to that target molecule. In some aspects,
the PCR primer sequence is removed from the end of each nucleic
acid target molecule. In some other aspects, the PCR primer
sequence is removed by digestion with a USER enzyme, followed by
end blunting with Klenow fragment polymerase and/or T4 DNA
polymerase.
[0124] In some aspects, amplified copies of the target molecules
are randomly fragmented to create molecules with a barcode sequence
at one end and a region of unknown sequence at the other end. In
some aspects, the fragmented nucleic acid molecules are
end-repaired to create blunt ends. In some aspects, biotinylated
nucleotides are incorporated into the repaired ends. In some
aspects, the fragmented nucleic acid molecules are circularized. In
some aspects, circularizing the fragmented molecules is carried out
by blunt-end ligation to bring the barcode sequence into proximity
with the unknown region of sequence from the interior of the
original target molecule. In some aspects, the circularized
molecules are fragmented to create linear molecules. In some
aspects, biotinylated molecules are attached to streptavidin-coated
beads to facilitate handling and purification. In some aspects, an
asymmetric adapter is ligated to each end of the linear
molecules.
[0125] In some aspects, adapter-ligated fragments are amplified or
copied. In some aspects, amplification is carried out by PCR using
two oligonucleotide primers, the first of which is complementary to
a constant sequence from the barcode-containing adapter, and the
second of which is complementary to the overhanging sequence of the
asymmetric adapter, and which together add sequences necessary for
sequencing.
Circularization and Fragmentation
[0126] In some aspects, fragmented nucleic acids are circularized.
Circularization of a nucleic acid can be carried out in any
suitable manner. In some aspects, circularization is carried out by
blunt-end ligation. In some aspects, this approach is used to
minimize the intervening sequence between the barcode sequence and
the unknown sequence region. In various aspects, sequencing such
intervening sequence(s) in every sequencing read wastes capacity
and decreases efficiency. In some aspects, the efficiency of
blunt-end ligation circularization is low, particularly for long
DNA molecules. In some aspects, circularization efficiency is
improved, including by the use of a bridging oligonucleotide or
adapter, by the creation of complementary sticky ends at the ends
of the fragment, or by the use of recombinases (Peng et al., PLoS
One 7(1): e29437, 2012).
[0127] In some aspects, a circularization adapter is used to
circularize fragmented PCR copies that already have been barcoded.
In some aspects, the circularized molecule is amplified by PCR. In
some aspects, the circularized molecule is amplified by RCA.
[0128] In some aspects, barcode-tagged fragments comprising the
barcode region at one end and a region of unknown sequence from an
interior portion of the target nucleotide sequence at the other end
are circularized, thereby bringing the barcode region into
proximity with the region of unknown sequence.
[0129] Fragmentation (or fragmenting) of nucleic acid molecules is
carried out in various aspects of the disclosure. For example, in
some aspects, the methods of the disclosure comprise multiple
fragmenting steps. Fragmenting of nucleic acids can be carried out
by any suitable method. In some aspects, the circularized,
barcode-tagged nucleic acid molecules are fragmented into linear
fragments, some of which contain barcodes.
[0130] In some aspects, fragmenting of the circularized molecules
is carried out by an acoustic shearing device (e.g., Covaris S2),
and/or by Nextera.TM. transposases (Epicentre, Madison, Wis.) to
combine shearing and the addition of asymmetric adapters. In some
aspects, transposase technology, such as that used in the
Nextera.TM. system (Epicentre), streamlines processing because
transposases simultaneously fragment DNA and introduce adapter
sequences at the newly exposed ends. Thus, transposases, in various
aspects, replace fragmentation or shearing, end repair, end
tailing, and adapter ligation with a single step. In some aspects,
therefore, transposases are used in fragmentation. For example, in
some aspects, transposes are used, e.g., for (1) fragmentation of
genomic or other extremely large DNA molecules into target
fragments 1-20 kb in length with concomitant attachment of
tripartite adapters; (2) fragmentation of long target fragments
with optional concomitant attachment of adapters designed to
improve circularization efficiency; and (3) fragmentation of
circularized DNA with concomitant attachment of asymmetric
adapters. Accordingly, in some aspects, transposases are used to
decrease the time necessary to prepare DNA samples for
sequencing.
Sequencing and Sequence Assembly
[0131] Various embodiments described herein relate to methods using
high-throughput sequencing. In some aspects, the term "bulk
sequencing," "massively parallel sequencing," or "next-generation
sequencing (NGS)" refers to any high-throughput sequencing
technology that parallelizes the DNA sequencing process. For
example, in some aspects, bulk sequencing methods are typically
capable of producing more than one million nucleic acid sequence
reads in a single assay. In some aspects, the terms "bulk
sequencing," "massively parallel sequencing," and "NGS" refer only
to general methods, not necessarily to the acquisition of greater
than one million sequence tags in a single run.
[0132] In some aspects, sequencing is carried out on any suitable
sequencing platform, such as reversible terminator chemistry (e.g.,
Illumina), pyrosequencing using polony emulsion droplets, e.g., 454
sequencing (e.g., Roche), ion semiconductor sequencing (Ion
Torrent.TM., Life Technologies), single molecule sequencing (e.g.,
SMRT, Pacific Biosciences, Menlo Park, Calif.), SOLiD sequencing
(Applied Biosystems), massively parallel signature sequencing, and
the like.
[0133] Various embodiments described herein relate to methods of
generating overlapping sequence reads and assembling them into a
contiguous nucleotide sequence ("contig") of a nucleic acid of
interest. In some aspects, assembly algorithms align and merge
overlapping sequence reads generated by methods described herein to
provide a contiguous sequence of a nucleic acid of interest. In
some aspects, nucleic acid sequence reads sharing the same barcode
sequences are identified and grouped. In some aspects, each group
of reads (i.e., grouped by a shared barcode sequence) is assembled
into one or more longer contiguous sequences.
[0134] In some aspects, grouping of sequences is carried out by a
computer program. For example, in various aspects, numerous
sequence assembly algorithms or sequence assemblers are utilized,
taking into account the type and complexity of the nucleic acid of
interest to be sequenced (e.g., genomic DNA, PCR product, plasmid,
and the like), the number and/or length of nucleic acids or other
overlapping regions generated, the type of sequencing methodology
performed, the read lengths generated, whether assembly is de novo
assembly of a previously unknown sequence or mapping assembly
against a reference sequence, and the like. In additional aspects,
an appropriate data analysis tool is selected based on the function
desired, such as alignment of sequence reads, base-calling and/or
polymorphism detection, de novo assembly, assembly from paired or
unpaired reads, or genome browsing and annotation.
[0135] In some aspects, overlapping sequence reads are assembled
into contigs or the full or partial contiguous sequence of the
nucleic acid of interest by sequence alignment, computationally or
manually, whether by pairwise alignment or multiple sequence
alignment of overlapping sequence reads.
[0136] In some aspects, overlapping sequence reads are assembled by
sequence assemblers including, but not limited to ABySS, AMOS,
Arachne WGA, CAP3, PCAP, Celera WGA Assembler/CABOG, CLC Genomics
Workbench, CodonCode Aligner, Euler, Euler-sr, Forge, Geneious,
MIRA, miraEST, NextGENe, Newbler, Phrap, TIGR Assembler,
Sequencher, SeqMan NGen, SHARCGS, SSAKE, Staden gap4 package,
VCAKE, Phusion assembler, Quality Value Guided SRA (QSRA), Velvet
(algorithm) (Zerbino et al., Genome Res. 18(5): 821-9, 2008),
SPAdes (http://bioinf.spbau.ru/spades), and the like.
[0137] In certain aspects, algorithms suited for short-read
sequence data may be used including, but not limited to, Cross
match, ELAND, Exonerate, MAQ, Mosaik, RMAP, SHRiMP, SOAP, SSAHA2,
SXOligoSearch, ALLPATHS, Edena, Euler-SR, SHARCGS, SHRAP, SSAKE,
VCAKE, Velvet, PyroBayes, PbShort, and ssahaSNP.
[0138] In some aspects, the methods provided herein provide for the
assembly of a contig or full continuous sequence of the nucleic
acid of interest at lengths in excess of about 1 kb, about 2 kb,
about 3 kb, about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8
kb, about 9 kb, about 10 kb, about 11 kb, about 12 kb, about 13 kb,
about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb,
about 19 kb, about 20 kb, about 25 kb, about 30 kb, about 35 kb,
about 40 kb, about 45 kb, or about 50 kb. In certain aspects, the
methods provided herein provide for the assembly of a target
nucleic acid with a length of about 0.1 kb, about 0.2 kb, about 0.3
kb, about 0.4 kb, about 0.5 kb, about 0.6 kb, about 0.7 kb, about
0.8 kb, about 0.9 kb, about 1.0 kb, about 1.1 kb, about 1.2 kb,
about 1.3 kb, about 1.4 kb, about 1.5 kb, about 1.6 kb, about 1.7
kb, about 1.8 kb, about 2.0 kb, about 2.1 kb, about 2.2 kb, about
2.3 kb, about 2.4 kb, about 2.5 kb, about 2.6 kb, about 2.7 kb,
about 2.8 kb, about 2.9 kb, about 3.0 kb, about 3.1 kb, about 3.2
kb, about 3.3 kb, about 3.4 kb, about 3.5 kb, about 3.6 kb, about
3.7 kb, about 3.8 kb, about 3.9 kb, about 4.0 kb, about 4.1 kb,
about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6
kb, about 4.7 kb, about 4.8 kb, about 4.9 kb, about 5.0 kb, about
5.2 kb, about 5.3 kb, about 5.4 kb, about 5.5 kb, about 5.6 kb,
about 5.7 kb, about 5.8 kb, about 5.9 kb, about 6.0 kb, about 6.1
kb, about 6.2 kb, about 6.3 kb, about 6.4 kb, about 6.5 kb, about
6.6 kb, about 6.7 kb, about 6.8 kb, about 6.9 kb, about 7.0 kb,
about 7.1 kb, about 7.2 kb, about 7.3 kb, about 7.4 kb, about 7.5
kb, about 7.6 kb, about 7.7 kb, about 7.8 kb, about 7.9 kb, about
8.0 kb, about 8.1 kb, about 8.2 kb, about 8.3 kb, about 8.4 kb,
about 8.5 kb, about 8.6 kb, about 8.7 kb, about 8.8 kb, about 8.9
kb, about 9.0 kb, about 9.1 kb, about 9.2 kb, about 9.3 kb, about
9.4 kb, about 9.5 kb, about 9.6 kb, about 9.7 kb, about 9.8 kb,
about 9.9 kb, about 10.0 kb, about 10.5 kb, about 11.0 kb, about
11.5 kb, about 12.0 kb, about 12.5 kb, about 13.0 kb, about 13.5
kb, about 14.0 kb, about 14.5 kb, about 15.0 kb, about 15.5 kb,
about 16.0 kb, about 16.5 kb, about 17.0 kb, about 17.5 kb, about
18.0 kb, about 18.5 kb, about 19.0 kb, about 19.5 kb, about 20.0
kb, about 20.5 kb, about 21.0 kb, about 21.5 kb, about 22.0 kb,
about 22.5 kb, about 23.0 kb, about 23.5 kb, about 24.0 kb, about
24.5 kb, about 25.0 kb, about 30.0 kb, about 35.0 kb, about 40.0
kb, about 45.0 kb, about 50.0 kb, about 55.0 kb, about 60.0 kb,
about 65.0 kb, about 70.0 kb, about 75.0 kb, about 80.0 kb, about
85.0 kb, about 90.0 kb, about 95.0 kb, or about 100 kb, or
greater.
[0139] Alternatively, in some aspects, the methods provided herein
provide for the assembly of a contig or full continuous sequence of
the nucleic acid of interest at lengths of less than about 1 kb,
about 900 bp, about 800 bp, about 700 bp, about 600 bp, or about
500 bp, or lesser.
[0140] In some aspects, the methods provided herein provide for the
assembly of a contig or full continuous sequence of the nucleic
acid of interest with very high per base accuracy or fidelity. The
term "accuracy" or "fidelity" as used herein refers to the degree
to which the measurement conforms to the correct, actual, or true
value of the measurement. For example, in some aspects, accuracy or
fidelity of the disclosed method is greater than about 80%, about
90%, about 95%, about 99%, about 99.5%, about 99.9%, about 99.95%,
about 99.99%, about 99.999%, or greater. In some aspects,
sequencing errors affecting per base and average accuracy of
sequence information due to the underlying sequencing platform are
substantially or completely corrected by majority calls by the
assembly methods and systems described herein, e.g., such as a
computer acting as an assembler. In some aspects, an output with a
single long read is produced from putting together multiple long
reads.
[0141] In particular aspects, the methods provided herein provide
for the assembly of the nucleic acid of interest with about 100%
accuracy, about 99.99% accuracy, about 99.98% accuracy, about
99.97% accuracy, about 99.96% accuracy, about 99.95% accuracy,
about 99.94% accuracy, about 99.93% accuracy, about 99.92%
accuracy, about 99.91% accuracy, about 99.90% accuracy, about
98.99% accuracy, about 98.98% accuracy, about 98.97% accuracy,
about 98.96% accuracy, about 98.95% accuracy, about 98.94%
accuracy, about 98.93% accuracy, about 98.92% accuracy, about
98.91% accuracy, about 98.90% accuracy, about 98.89% accuracy,
about 98.88% accuracy, about 98.87% accuracy, about 98.86%
accuracy, about 98.85% accuracy, about 98.84% accuracy, about
98.83% accuracy, about 98.82% accuracy, about 98.81% accuracy,
about 98.80% accuracy, about 98.79% accuracy, about 98.78%
accuracy, about 98.77% accuracy, about 98.76% accuracy, about
98.75% accuracy, about 98.74% accuracy, about 98.73% accuracy,
about 98.72% accuracy, about 98.71% accuracy, about 98.70%
accuracy, about 98.69% accuracy, about 98.68% accuracy, about
98.67% accuracy, about 98.66% accuracy, about 98.65% accuracy,
about 98.64% accuracy, about 98.63% accuracy, about 98.62%
accuracy, about 98.61% accuracy, about 98.60% accuracy, about 98.5%
accuracy, about 98.0% accuracy, about 97.5% accuracy, about 97.0%
accuracy, about 96.5% accuracy, about 96.0% accuracy, about 95.5%
accuracy, about 95.0% accuracy, about 94.5% accuracy, about 94.0%
accuracy, about 93.5% accuracy, about 93.0% accuracy, about 92.5%
accuracy, about 92.0% accuracy, about 91.5% accuracy, about 91.0%
accuracy, about 90.5% accuracy, about 90.0% accuracy, about 89.%
accuracy, about 88% accuracy, about 87% accuracy, about 86%
accuracy, about 85% accuracy, about 84% accuracy, about 83%
accuracy, about 82% accuracy, about 81% accuracy, or about 80%
accuracy.
[0142] In some aspects, the methods provided herein provide for the
assembly of a contig or full continuous sequence of the nucleic
acid of interest with an error rate of about 0.001%, about 0.002%,
about 0.003%, about 0.004%, about 0.005%, about 0.006%, about
0.007%, about 0.008%, about 0.009%, about 0.010%, about 0.011%,
about 0.012%, about 0.013%, about 0.014%, about 0.015%, about
0.016%, about 0.017%, about 0.018%, about 0.019%, about 0.020%,
about 0.025%, about 0.030%, about 0.035%, about 0.040%, about
0.045%, about 0.050%, about 0.055%, about 0.060%, about 0.065%,
about 0.070%, about 0.075%, about 0.080%, about 0.085%, about
0.090%, about 0.095%, about 0.10%, about 0.15%, about 0.20%, about
0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, about
0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about
0.75%, about 0.80%, about 0.85%, about 0.90%, about 0.95%, about
1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%,
about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about
2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%,
about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about
3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%,
about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about
4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%,
about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about
7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%,
about 10.0%, about 15%, or about 20%.
[0143] In some aspects, the methods described herein take less than
5 days, less than 4 days, less than 3 days, less than 2 days, or
less than 1 day. In particular aspects, methods described herein
take about 3 days, because the methods comprise elements that run
overnight (i.e., PCR amplification and ligation). In some aspects,
methods are shortened (or sped up) by the use of faster PCR
thermocyclers and faster polymerases, and/or by using higher
concentrations of ligase. Such improvements, in some aspects,
shorten the protocol to two days. Further improvements, including
the use of Nextera.TM. transposon, as described herein above, also
eliminates protocol components, speeds up the protocol, and
shortens overall method time.
[0144] In some aspects, the methods described herein are much
simpler and more convenient than other methods. For example, in
some aspects, the methods of the disclosure are carried out in a
single tube, thus involving less handling, and eliminating the need
to split the library into multiple-well plates.
[0145] In some aspects, the methods of the disclosure facilitate
haplotyping of chromosomes of polyploid species. A haplotype is a
collection of specific alleles (e.g., particular DNA sequences) in
a cluster of tightly-linked genes on a chromosome that are likely
to be inherited together. In other words, a haplotype is the group
of genes that a progeny inherits from one parent. A cell or a
species is "polyploid" if it contains more than two haploid (n)
sets of chromosomes. In other words, the chromosome number for the
cell or species is some multiple of n greater than the 2n content
of diploid cells. For example, triploid (3n) and tetraploid cell
(4n) cells are polyploid. In some aspects, the methods of the
disclosure are useful in haplotype reconstruction from sequence
data, or by haplotype assembly. Methods of the Disclosure
[0146] For example, in one example embodiment, fragments of nucleic
acid are assembled into distinct nucleic acid sequences by
fragmenting a target nucleic acid molecule and attaching the same
random nucleic acid barcode to each short sequencing-ready nucleic
acid fragment that derives from the nucleic acid molecule. To each
end of each fragment in the starting library is ligated a first
"tripartite" adapter comprising an outer PCR annealing region, a
central random barcode sequence, and an inner sequencing primer
region. The adapter-ligated library is then diluted, and about one
million molecules (or more or fewer) are amplified by PCR using a
primer complementary to the PCR annealing region on the adapter. In
various aspects, the library is diluted by orders of magnitude
greater or lesser than the million molecules, depending on the goal
of the sequencing and the resources available. For example, the
complexity depends upon the amount of sequencing and the length of
the target. In some aspects, about 10,000 or more molecules are
amplified; whereas, in some aspects about 1,000,000 or more
molecules are amplified. In some aspects, dilution of the library
ensures that enough reads are derived from each molecule to allow
full assembly. Each of the about one million library sequences is
copied many times with PCR. The PCR annealing region is removed
from each 5' end of the amplified nucleic acid with USER.TM.
enzyme, which cuts the DNA backbone at uracil bases designed into
the PCR primer. The barcode sequences are thus positioned at the
ends of each molecule. An enzyme mixture called dsDNA fragmentase
is then used to randomly cut each copy in a different location. The
ends of the nucleic acid are repaired (blunted) in the presence of
biotin-dCTP, which results in biotinylation of the ends of the
nucleic acid molecules. In some aspects, dC nucleotides are
designed into the tripartite adapter to ensure successful
biotinylation. The nucleic acid is then circularized, bringing the
barcode sequence at one end into proximity with an unknown sequence
region randomly selected from the length of the starting molecule.
The circularized nucleic acid is again fragmented, this time by
shearing (including, in some aspects, mechanical or acoustic
shearing), to obtain molecules of a desired length. In some
aspects, the desired nucleic acid length is about 300 bp to about
800 bp, but this may be modified depending on the sequencing
instrument used and the goals of the sequencing. In some aspects,
the nucleic acid fragments containing the barcodes are bound to
streptavidin-coated magnetic beads, end-repaired, dA-tailed, and
ligated to another adapter. This "second" adapter comprises two
oligonucleotides of different lengths, such that when annealed the
shorter oligonucleotide has a 3' dT overhang and the longer
oligonucleotide, which corresponds to a second sequencing primer
annealing sequence, has a longer 3' overhang. In some aspects, only
the longer oligonucleotide (and not the subsequently synthesized
reverse complement of the shorter adapter) is able to subsequently
anneal to the PCR primer. The beads are added to a PCR mixture
containing primers that anneal to the two sequencing primer regions
(one of which was added by the first adapter, the other by the
second adapter). PCR exponentially amplifies only the region of the
template from the first sequencing primer, in the direction of the
barcode and the sequence of interest, through the second adapter,
and adds sequences that allow annealing to the sequencing flow
cell. In some aspects, the resulting nucleic acid molecules are
size-selected. In some aspects, size selection and, therefore,
tighter size distribution leads to better sequencing results.
[0147] If size selected, the size selection is carried out by the
Agencourt AMPure XP system (Beckman Coulter, Brea, Calif.), or by
gel purification. The nucleic acid molecules are then sequenced,
using a single-end read or paired-end reads. The sequencing data
from the first read contains the barcode sequence followed by
sequence from the original fragment. In some aspects, it also is
possible to switch the method so that the barcode is on the second
read. All sequences with identical barcodes are grouped, and each
group is assembled into the full-length sequence independent of the
others. In various aspects, this method is adapted for use on any
of the available high-throughput sequencing platforms.
[0148] In a further aspect, the embodiment outlined above generates
two barcode-defined groups of reads corresponding to each original
target molecule, defined by the two distinct barcode sequences in
the adapters that are ligated to the two ends of the target
molecule. Each target molecule is thus "tagged" with two different
barcode sequences. Fragments containing one of the two barcode
sequences are pooled and assembled separately from those containing
the other barcode sequence. In some aspects, the two barcode
sequences are linked by a supplemental experimental preparation
and/or computational analysis, allowing all reads containing either
of the barcode sequences to be pooled and assembled together. In
some aspects, the length of the target molecules that are sequenced
is thereby doubled, the efficiency of the method is increased, and
the problem of decreasing circularization efficiency with
increasing molecule length is partially offset. In some aspects, a
subset of the PCR-amplified, barcode adapter-ligated target
molecules is not fragmented. In some aspects, a subset is
physically separated from the fragmented population, and this
separated fraction is not subjected to fragmentation. In other
aspects, fragmentation of the population is incomplete, and those
molecules that escape fragmentation are used for barcode linking.
In some aspects, circularization of intact molecules brings the two
barcode sequences ligated to that target molecule into proximity.
In some aspects, the region containing the two barcode sequences is
separated from the target molecule by PCR or restriction
endonuclease digestion, converted into sequencing-ready molecules
by the addition of appropriate adapter sequences, and sequenced in
the same sequencing run as the main library or in a separate run.
In the bioinformatic processing pipeline, these linked barcode
sequence pairs are identified, and groups of reads tagged with each
of the barcode sequences are merged into a single group for
assembly into the longer sequence.
[0149] In some aspects of the methods described herein, barcode
sequences are linked. In some aspects, the linked barcode sequences
allow the two barcode-defined groups of reads to be merged by
circularizing a small percentage of the products of the first PCR
amplification while forgoing fragmentation, such that the barcode
sequences at each end are brought into proximity with one another.
In some aspects, the circularized full-length molecules remain in
the same mixture as the circularized fragmented molecules. In some
aspects, both types of molecule are processed together and
sequenced in the same sequencing reaction. In various aspects,
sequencing reads capturing paired barcode sequences are identified
computationally. In some aspects, when this approach is used, it is
desirable to use a mixture of tripartite adapters containing
distinct sequencing primer regions to avoid hairpin formation.
Alternatively, forked adapters are used so that the two ends of the
target molecules receive different sequencing primer sequences. In
some aspects, a portion of the circularized mixture is removed
(before or after fragmentation) and used to prepare samples for
barcode pairing. In some aspects, the circularized molecules (which
may or may not have previously been fragmented to open the circles)
are digested with a restriction endonuclease that recognizes a
specific site in the constant regions of the barcode adapter (e.g.,
in one aspect, the restriction endonuclease SapI recognizes a site
in the sequence of the Illumina TruSeq adapter sequence). In some
aspects, asymmetric adapters are ligated to the newly exposed
sticky end or ends. In some aspects, the adapter-ligated fragments
are amplified by PCR using two oligonucleotide primers, the first
of which is complementary to a constant sequence from the
barcode-containing adapter, and the second of which is
complementary to the overhanging sequence of the asymmetric
adapter, and which together add sequences for sequencing on a
sequencing instrument (e.g., Illumina). In some aspects, forked or
Y-shaped adapters are ligated to the newly exposed end or ends. In
some aspects, the adapter-ligated fragments are amplified by PCR
using two oligonucleotide primers, one of which is complementary to
a sequence on one fork of the adapter and the other of which is
complementary to a sequence on the second fork of the adapter. The
type of adapters used depends on what barcode adapter design is
used. In some aspects, the two barcode sequences are identified in
the sequencing data. In some aspects, the two groups of reads in
the primary sequencing data set defined by each of the linked
barcodes are merged and assembled into longer sequences. In most
aspects, the short constant sequences bordering the barcodes
identify true barcode pairs from spurious sequences.
[0150] In a particular aspect, the disclosure provides a method for
obtaining nucleic acid sequence information from a nucleic acid
molecule by assembling a series of short nucleic acid sequences
into longer nucleic acid sequences (i.e. intermediate or long
nucleic acid sequences). In most aspects, the method comprises
some, if not all, of fragmenting the nucleic acid molecule
comprising a nucleic acid sequence or a genomic nucleic acid
sequence into a plurality of linear nucleic acid sequences;
attaching a first adapter to the linear nucleic acid sequence, the
first adapter comprising an outer polymerase chain reaction (PCR)
primer region (or nucleic acid amplification region), an inner
sequencing primer region, and a central barcode region to each end
of the linear nucleic acid sequences to form barcode-tagged
sequences, wherein the first adapter attached at one end comprises
a different barcode than the first adapter attached at the other
end; replicating the barcode-tagged sequences, e.g., by PCR, to
obtain a library of barcode-tagged sequences using a primer
complementary to the PCR primer region; removing the PCR primer
region from the barcode-tagged sequences; breaking the
barcode-tagged sequences at random locations using an enzyme that
generates linear, barcode-tagged fragments comprising the barcode
region at one end and a region of unknown sequence at the other
end; circularizing the linear, barcode-tagged fragments comprising
the barcode region at one end and a region of unknown sequence from
an interior portion of the target nucleotide sequence at the other
end, thereby bringing the barcode region into proximity with the
region of unknown sequence; fragmenting the circularized,
barcode-tagged fragments into linear, barcode-tagged fragments;
attaching a second adapter comprising two oligonucleotides of
different lengths to each end of the linear, barcode-tagged
fragments to form double adapter-ligated barcode-tagged nucleic
acid fragments, wherein one end of the second adapter is double
stranded to facilitate ligation and the other end of the second
adapter comprises a 3' single-stranded overhang, and wherein only
the longer of the two oligonucleotides comprises a sequence
complementary to a second sequencing primer and comprises
sufficient length to allow annealing of that primer; replicating
the double adapter-ligated barcode-tagged nucleic acid fragments by
PCR using two primers, the first of which is complementary to a
constant sequence from the barcode-containing adapter, and the
second of which is complementary to the overhanging sequence of the
asymmetric adapter, and which together add sequences necessary for
nucleic acid sequencing; sequencing the double adapter-ligated
barcode-tagged nucleic acid fragments beginning with the barcode
region followed by the target sequence; sorting a series of
sequenced nucleic acid fragments into independent groups based on
shared barcodes; and assembling each group of short nucleic acids
into one or more longer nucleic acid sequences, independent of all
other groups.
[0151] Sample Preparation
[0152] In some example aspects of the disclosure, nucleic acid
samples are prepared as described below. Only one strand of the
nucleic acid is described and set out below.
[0153] (1) A tripartite adapter is ligated to the end of the target
molecule:
TABLE-US-00001 Ligated target-
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNCCAGGAAT
AGTTATGTGCATTAATGAATGGCGCC
[0154] (2) Target molecules with adapters at both ends are
amplified and the PCR primer annealing region (i.e., the region
after " . . . NNNNCC") is removed:
TABLE-US-00002 Ligated target-
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNCC
[0155] (3) Amplified target molecules are fragmented and
circularized:
TABLE-US-00003 Ligated target end-
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNCC- ligated region of
interest
[0156] (4) Circularized DNA is fragmented and fragments containing
adapter sequences are prepared for sequencing:
TABLE-US-00004 Adapter 1 (e.g., Illumina)-
CCTACACGACGCTCTTCCGATCNNNNNNNNNNNNNNNNCC- ligated region of
interest-Adapter 2 (e.g., Illumina)
[0157] (5) Resulting sequencing read:
TABLE-US-00005 NNNNNNNNNNNNNNNNCC - ligated region of interest
[0158] (6) In the computational pipeline, the sequences at the
start of the read are used to determine the sample and target
molecule of origin:
TABLE-US-00006 NNNNNNNNNNNNNNNNCC-ligated region of interest
[0159] The 5' multiple N region determines the target molecule of
origin. The "CC" region confirms the upstream sequence is a
barcode. The 3' region contains sequence information for the
ligated region of interest.
Sample Preparation for Barcode Pairing
[0160] In some aspects, samples are prepared for barcode pairing as
described below. Only one strand of the nucleic acid is described
and set out below.
[0161] (1) Tripartite adapter is ligated to the end of the target
molecule:
TABLE-US-00007 Ligated target-
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNCCAGGAAT
AGTTATGTGCATTAATGAATGGCGCC
[0162] (2) Target molecules with adapters at both ends are
amplified and the PCR primer annealing region (i.e., the region
after " . . . NNNNCC"):
TABLE-US-00008 Ligated target-
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNCC
[0163] (3) Full-length amplified target molecules that avoid
fragmentation are circularized:
TABLE-US-00009 Ligated target end-
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNCC-
GGNNNNNNNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGGNNN- Ligated target
end
[0164] (4) Circularized DNA is fragmented and fragments containing
adapter sequences are prepared for sequencing:
TABLE-US-00010 Adapter 1 (e.g. Illumina)-NNNNNNNNNNNNNNNNCC-
GG-NNNNNNNNNNNNNNNN-Adapter 2 (e.g., Illumina)
[0165] (5) Resulting sequencing read:
TABLE-US-00011 NNNNNNNNNNNNNNNNCC-GGNNNNNNNNNNNNNNNN-Adapter 2
(e.g., Illumina)
[0166] (6) In the computational pipeline, the two barcodes (i.e.,
the multiple "Ns" set out below at each end of the sequence) are
identified as a pair:
TABLE-US-00012 NNNNNNNNNNNNNNNNCC - GGNNNNNNNNNNNNNNNN
[0167] The 5' and 3' multiple N regions represent the paired
barcodes.
Multiplexed Sample Preparation
[0168] In some aspects, multiplexed samples are prepared as
described below. Only one strand of the nucleic acid is described
and set out below.
[0169] (1) Tripartite adapter is ligated to the end of the target
molecule. Underlined, bolded font indicates the index sequence
(e.g., ) unique to each sample:
TABLE-US-00013 Ligated target -
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNN CAG
GAATAGTTATGTGCATTAATGAATGGCGCC
[0170] (2) Target molecules with adapters at both ends are
amplified and the PCR primer annealing region (i.e., the region
after " . . . ") is removed:
TABLE-US-00014 Ligated target-
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNN C
[0171] (3) PCR products deriving from multiple samples are mixed
and processed together in a single tube from this point. Each
contains a unique index sequence. Amplified target molecules are
fragmented and circularized:
TABLE-US-00015 Ligated target end -
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNN C - ligated region of
interest
[0172] (4) Circularized DNA is fragmented and fragments containing
adapter sequences are prepared for sequencing:
TABLE-US-00016 Adapter 1 (e.g., Illumina)
CCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNN C - ligated region of
interest-Adapter 2 (e.g., Illumina)
[0173] (5) Resulting sequencing read:
TABLE-US-00017 NNNNNNNNNNNNNNNN C - ligated region of interest
[0174] (6) In the computational pipeline, the sequences at the
start of the read are used to determine the sample and target
molecule of origin:
TABLE-US-00018 NNNNNNNNNNNNNNNN C -ligated region of interest
[0175] The 5' N region represents the barcode and determines the
origin of the target molecule. The "" region represents the index
sequence and determines origin of the sample. The ligated region of
interest contains the sequence information.
Multiplexed Sample Preparation for Barcode Pairing
[0176] In some aspects, multiplexed samples are prepared for
barcode pairing as described below. Only one strand of the nucleic
acid is described and set out below.
[0177] (1) Tripartite adapter is ligated to the end of the target
molecule. Underlined, bolded font indicates the index sequence
(e.g., ATCACG) unique to each sample:
TABLE-US-00019 Ligated target -
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNN CAG
GAATAGTTATGTGCATTAATGAATGGCGCC
[0178] (2) Target molecules with adapters at both ends are
amplified and the PCR primer annealing region (i.e., the region
after " . . . NNC") is removed:
TABLE-US-00020 Ligated target - NNNCCTACACGACGCTCTTCCGATCTNNNNNNNN
NNNNNNNN C
[0179] (3) PCR products deriving from multiple samples are mixed
and processed together in a single tube from this point. Each
contains a unique index sequence (underlined font). Full-length
amplified target molecules that avoid fragmentation are
circularized:
TABLE-US-00021 Ligated target end -
NNNCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNN C - G
NNNNNNNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGGNNN - Ligated target end
[0180] (4) Circularized DNA is fragmented and fragments containing
adapter sequences are prepared for sequencing:
TABLE-US-00022 Adapter 1 (e.g., Illumina) - NNNNNNNNNNNNNNNN C -
GCGTGATNNNNNNNNNNNNNNNN -Adapter 2 (e.g., Illumina)
[0181] (5) Resulting sequencing read:
TABLE-US-00023 NNNNNNNNNNNNNNNN C -G NNNNNNNNNNNNNNNN- Adapter 2
(e.g., Illumina)
[0182] (6) In the computational pipeline, the two barcodes are
identified as a pair and the index determines the sample of origin.
Matching indexes confirm intramolecular circularization:
TABLE-US-00024 NNNNNNNNNNNNNNNN C -G NNNNNNNNNNNNNNNN
[0183] The 5' and 3' multiple N regions represent the paired
barcodes. The "" region represents the index sequence and
determines origin of sample. The "" sequence or region is the
reverse complement of the first index sequence, confirming
intramolecular circularization.
Computational Pipeline and Sequence Assembly
[0184] In some aspects, once a library created according to the
methods of the disclosure has been sequenced, the sequencing data
is processed to assemble the raw short nucleic acid sequences (or
short reads) into synthetic long nucleic acid sequences (long
reads). The "computational pipeline" or "processing pipeline" is as
described below.
[0185] In some aspects, sequencing reads are trimmed to remove
regions of low quality, as well as known adapter sequences. A
number of open-source tools are available for this purpose
including, but not limited to, Trimmomatic
(http://www.usadellab.org/cms/?page=trimmomatic), Skewer
(http://www.biomedcentral.com/1471-2105/15/182), the FASTX-toolkit
(http://hannonlab.cshl.edu/fastx toolkit/), Scythe
(http://github.com/vsbuffalo/scythe), and others.
[0186] In some aspects, sequencing reads are searched for barcode
sequences. In some aspects, the first sixteen bases of the read are
identified as a barcode if the subsequent bases match the known
constant region in the tripartite adapter, e.g., "CC." The barcode
sequence, the constant sequence, and any other adapter sequences
(such as sequences left over from incomplete removal of the PCR
primer region) are removed from the read. The remainder of the read
constitutes sequence information from the molecule identified by
the specific barcode. In some aspects, a hash table is created in
which the barcode sequences are the keys and the sequence
information is the values. That is, each distinct barcode defines a
bin, and each sequence read is placed in the bin defined by its
barcode. In some aspects, if paired-end reads are used, the reverse
read is placed in the same bin as the forward read.
[0187] In some aspects, when barcode pairing data is available,
those reads are analyzed to find paired barcodes. After trimming
adapters and low-quality regions, reads are inspected for the
expected pattern, e.g., barcode 1, defined sequence 1, reverse
complement of defined sequence 2, reverse complement of barcode 2,
and adapter sequence. Barcode pairs are extracted from sequences
matching this pattern. In some aspects, a data structure is created
to count how many times each barcode is paired with other barcodes.
A true pair is verified when two barcodes are paired with each
other more times than a threshold number and more times than either
is paired with any other barcode. Once a true pair is verified, the
sequence read bins corresponding to the two barcodes are merged
into a single bin for assembly.
[0188] In some aspects, the sequences in each barcode-defined bin
are assembled into synthetic long reads. Each bin is assembled
independently of the other bins, allowing parallelization of
assembly. A number of open-source assemblers are available and are
described herein above.
[0189] In one aspect, the present disclosure includes a
computational pipeline for assembling grouped reads. After quality
checking each read for low-confidence calls and for sequences
matching the adapters used in the protocol, the first bases can be
split from the read and defined as the barcode. In some
embodiments, a hash table is built that groups the subset of reads
associated with each barcode. In some embodiments, each group is
then assembled individually, with or without a reference genome,
using standard alignment and assembly software (e.g., Bowtie 2,
Velvet, or SPAdes).
[0190] In some embodiments, the method is used with nanopore
sequencing platforms as described in U.S. Patent Publication Number
2014/0034497, which is herein incorporated by reference in its
entirety. In some embodiments, the method is used with Pacific
Biosciences sequencing platforms as described in U.S. Pat. Nos.
7,315,019 and 8,652,779, which are each herein incorporated by
reference in their entirety. In some embodiments, the method is
used with Illumina sequencing platforms as described in U.S. Pat.
No. 7,115,400 and PCT Publication Number WO/2007/010252, which are
herein incorporated by reference in their entirety. In some
embodiments, the method is used with IonTorrent sequencing
platforms as described in PCT Patent Publication Number
WO/2008/076406, which is herein incorporated by reference in its
entirety. In some embodiments, the method is used with Roche/454
sequencing platforms as described in U.S. Patent Number
WO/2004/070005, which is herein incorporated by reference in its
entirety.
[0191] In some embodiments, for which data is provided below as
examples, the method comprises the operations of: (a) creating a
target nucleic acid library (e.g., by mechanical shearing, PCR,
restriction digestion, or another method); (b) preparing that
library for adapter attachment (e.g., by end-repair and
dT-tailing); (c) creating a mixture of adapter fragments (e.g.,
comprising regions that are identical among all members of the
adapter population and a degenerate "barcode" region that is unique
to each member of the population); (d) attaching one adapter to
each end of each member of the target library (e.g., by ligation);
(e) amplifying the adapter-ligated target molecules by PCR (e.g.,
using a single, uracil-containing oligonucleotide primer that is
complementary to a constant region of the adapters lying 5' of the
barcode sequence, to create many copies of each target molecule
such that each copy of the same target molecule is attached to the
same barcode sequences unique to that target molecule); (f)
optionally removing the PCR primer sequence from the 5' end of each
DNA strand (e.g., by digestion with USER enzyme); (g) randomly
fragmenting the amplified copies of the targets (e.g., to create
molecules with a barcode sequence at one end and a region of
unknown sequence at the other end); (h) end-repairing the
fragmented molecules (e.g., to create blunt ends while
incorporating biotinylated nucleotides into the repaired ends); (i)
circularizing the fragmented molecules (e.g., by blunt-end ligation
to bring the barcode sequence into proximity with the unknown
region of sequence from the interior of the original target
molecule); (j) fragmenting the circularized molecules (e.g., to
create linear molecules); (k) optionally attaching the biotinylated
molecules to streptavidin-coated beads (e.g., to facilitate
handling and purification); (1) ligating an asymmetric adapter to
each end of the linear molecules; (m) amplifying the
adapter-ligated fragments (e.g., by PCR using two oligonucleotide
primers, the first of which is complementary to a constant sequence
from the barcode-containing adapter, and the second of which is
complementary to the overhanging sequence of the asymmetric
adapter, and which together add sequences necessary for sequencing
on a sequencing instrument); (n) sequencing the amplified DNA
(e.g., on a massively parallel short-read instrument); (o)
computationally identifying and grouping reads sharing the same
barcode sequences; and (p) assembling each group of reads (e.g.,
defined by a shared barcode sequence into longer contiguous
sequences describing the original target molecule).
[0192] The embodiment outlined above can generate two
barcode-defined groups of reads corresponding to each original
target molecule (e.g., defined by the two distinct barcode
sequences in the adapters that ligated to the two ends of the
target molecule). Each target molecule can be tagged with two
different barcode sequences. Fragments containing one of the two
barcode sequences can be pooled and assembled separately from those
containing the other barcode sequence. In some embodiments the two
barcode sequences are linked by a supplemental experimental
preparation, allowing all reads containing either of the barcode
sequences to be pooled and assembled together. In some embodiments,
a subset of the PCR-amplified, barcode adapter-ligated target
molecules is not fragmented. In some embodiments, this subset is
physically separated from the fragmented population, and this
separated fraction is not subjected to fragmentation. In some
embodiments, fragmentation of the population is incomplete, and
those molecules that escape fragmentation are used for barcode
linking. Circularization of intact molecules brings the two barcode
sequences ligated to that target molecule into proximity. In some
embodiments, the region containing the two barcode sequences is
separated from the target molecule (for example, by PCR or
restriction endonuclease digestion), converted into
sequencing-ready molecules by the addition of appropriate adapter
sequences, and sequenced in the same sequencing run as the main
library or in a separate run. In some embodiments, during the
bioinformatic processing pipeline, these linked barcode sequence
pairs are identified, and groups of reads tagged with each of the
barcode sequences are merged into a single group for assembly.
[0193] In some embodiments, barcode sequences can be linked as
follows: (a) circularizing (a small percentage of) the products of
the first PCR amplification while forgoing the fragmentation (e.g.,
such that the barcode sequences at each end are brought into
proximity with one another); (b) digesting the circularized
molecules (e.g., with a restriction endonuclease that recognizes a
specific site in the constant regions of the barcode adapter (in a
some embodiments, the restriction endonuclease SapI recognizes a
site in the sequence of the Illumina TruSeq adapter sequences));
(c) ligating asymmetric adapters to the newly exposed sticky end or
ends; (d) amplifying the adapter-ligated fragments (e.g., by PCR
using two oligonucleotide primers, the first of which is
complementary to a constant sequence from the barcode-containing
adapter, and the second of which is complementary to the
overhanging sequence of the asymmetric adapter, and which together
add sequences necessary for sequencing on a sequencing instrument);
(e) sequencing the amplified DNA (e.g., on a massively parallel
short-read instrument); (f) identifying the two barcode sequences
in the sequencing data; and (g) merging the two groups of reads in
the primary sequencing data set defined by each of the linked
barcodes (e.g., and assembling them together into longer sequences
describing the target molecule that barcode adapters containing the
two linked barcode sequences were ligated).
[0194] In some embodiments, as an alternative to downstream linkage
of two distinct barcode sequences ligated to the two ends of the
target molecule, both ends of the target molecule are tagged with
the same barcode sequence. A single circularization barcode adapter
can be ligated to the target molecule in lieu of two end adapters.
The two ends of this adapter can ligate to the two ends of the same
target molecule to form a circular molecule.
[0195] Methods that attach the same barcode sequence to both ends
of the target molecule via circularization, including those
described herein, have advantages that include: (1) target
molecules that escape barcoding can be removed by exonucleases on
the basis of remaining linear; and (2) barcoded molecules can be
quantified by quantitative PCR (qPCR) by amplifying a short (e.g.,
50-100 bp) amplicon corresponding to sequences within the
circularization adapter, rather than needing to amplify the entire
target molecule.
[0196] In some embodiments, the adapter contains a single barcode
sequence. In some embodiments, the barcode sequence is flanked in
the 5' direction on each strand by uracil bases. After
circularization, enzymes (for example, the USER enzyme mix (New
England Biolabs)) can excise the uracils and break the phosphate
backbone. Each strand can be broken in the 5' direction of the
barcode sequence, opening the circular molecule into a linear
molecule with 5' single-stranded overhangs at each end that contain
the same barcode sequence. In some embodiments, enzymatic extension
of the 3' ends (for example, by Klenow exo-DNA polymerase or Taq
DNA polymerase) copies the barcode sequence at each end, creating a
fully double-stranded DNA molecule with the same barcode sequence
at both ends. In some embodiments, extension by appropriate DNA
polymerase enzymes leaves dA-tails useful for ligating additional
adapters containing sequences that serve as PCR primer annealing
sites for subsequent PCR amplification.
[0197] In some embodiments, the circularization adapter is prepared
prior to ligation such that it contains two copies of the barcode
sequence, or one copy of the barcode sequence and another copy of
the reverse complement of that barcode sequence. In some
embodiments, following circularization, the adapter is cut between
the two barcodes prior to amplification. It can be advantageous to
circularize the target around the barcode adapter such that the
same barcode sequence becomes associated with both ends of the
target molecule.
[0198] In some embodiments, adapters are attached by ligation. In
some embodiments, ligation is facilitated by single-nucleotide
tailing. In some embodiments, the adapters are dA-tailed and the
targets are dT-tailed. In some embodiments, the adapters are
dT-tailed and the targets are dA-tailed. In some embodiments,
adapters are attached by blunt-end ligation. In some embodiments,
adapters are incorporated during amplification. In some
embodiments, adapter sequences are contained within PCR
primers.
[0199] In some embodiments, interior regions of the amplified
target molecule are exposed prior to circularization by
fragmentation. In some embodiments, fragmentation is performed
using the dsDNA fragmentase enzyme mixture from New England
Biolabs, a mixture of two enzymes that creates random breaks in
double-stranded DNA. Unlike exonucleases, fragmentase preserves
both ends of the DNA molecule, both of which can give rise to
productive circular molecules; unlike mechanical shearing, breaks
are introduced along the length of the DNA molecule independent of
the distance from an end or the size of the molecule; and the
number of breaks per kilobase can be adjusted for different target
molecule lengths by diluting the enzyme mixture or adjusting the
reaction time. In some embodiments, fragmentation is achieved by
mechanical shearing, or concatamerization by ligation followed by
shearing.
[0200] In some embodiments, in place of mechanical or enzymatic
fragmentation, fragments with random ends are generated during
amplification with random (degenerate) or partially random
oligonucleotide primers. In some embodiments, amplification is
followed by further amplification with non-random primers. In some
embodiments, amplification is followed by restriction digestion or
other enzymatic treatments. In some embodiments, fragments with
random ends are generated as described below in Example 8.
[0201] In some embodiments, barcode adapter-ligated target
molecules are amplified with PCR. In some embodiments, the PfuCx
Turbo DNA polymerase (Agilent) is used for PCR. This enzyme is
compatible with uracil-containing primers, yet features a
proofreading activity that reduces the error rate relative to Taq
polymerases. In some embodiments, a single primer is used for PCR.
Using a single primer has been shown to discourage the accumulation
of primer dimers during PCR. In some embodiments, two or more
distinct primers are used for PCR.
[0202] In some embodiments, the PCR mixture is supplemented with
betaine, DMSO, or other additives or combinations thereof to reduce
the sequence dependence of amplification efficiency, promoting a
more even distribution of amplified products.
[0203] In some embodiments, the adapters that are attached to the
two ends of a target molecule are identical. In some embodiments,
the adapters that are attached to the two ends of a target molecule
are distinct. In some embodiments, the adapters incorporate
distinct PCR primer-annealing sequences and/or distinct sequencing
primer-annealing sequences into the two ends of the target
molecule. In some embodiments this is accomplished by adding a
mixture of different adapters into the ligation mixture. In some
embodiments a "forked" or "Y" adapter is used, comprising two
oligonucleotides that are only partially complimentary, such that
they anneal to form an adapter that is double stranded and ligation
competent at one end, but forks into two non-complimentary single
strands at the other end.
[0204] In some embodiments, amplification bias is reduced by a
linear amplification stage prior to exponential amplification. In
some embodiments, barcode-containing adapters with 3' overhangs are
attached to the ends of the target molecule, such that only one of
the two strands of the ligated target molecule is capable of
annealing to a PCR primer at a set annealing temperature. In some
embodiments, exponential amplification is triggered by the addition
of a nested primer. In some embodiments, exponential amplification
is triggered by a change in the annealing temperature.
[0205] In some embodiments, amplification is achieved by
rolling-circle amplification (RCA) or hyperbranching rolling-circle
amplification (HRCA). In some embodiments, a circularization
adapter is ligated to the target, such that the two ends of the
adapter ligate to the ends of the same target molecule to form a
circular molecule. In some embodiments, the adapter contains a
single barcode sequence, which is flanked in the 5' direction on
each strand by nicking endonuclease recognition sequences. In some
embodiments, the double-stranded DNA concatamers that result from
RCA or HRCA are broken, by, for example, mechanical shearing or
dsDNA fragmentase. In some embodiments, the resulting fragments are
further treated with the nicking endonuclease, which introduce
single-strand breaks on each side of the barcode, so that each
strand of the barcode section becomes a 5' overhang at the end of
the resulting fragments. In some embodiments, Klenow or another
polymerase fills in these ends, copying the barcode to create a
blunt end ready for circularization. In some embodiments, two loop
adapters are ligated to the ends of the target to create a circular
"dumbbell" structure that can be amplified by RCA or HRCA. In some
embodiments, the resulting concatamers are fragmented and digested
by a nicking endonuclease as described herein.
[0206] In some embodiments, some or all of the amplification is
performed within emulsified compartments.
[0207] In some embodiments, fragmented PCR products are
circularized by blunt-end ligation. In some embodiments, fragmented
molecules are circularized with a bridging oligonucleotide or
adapter, the creation of complementary sticky ends at the ends of
the fragment, or the use of recombinases.
[0208] In some embodiments, short defined sequences are designed to
follow the barcode sequence in the sequencing reads to positively
distinguish true barcode sequences from spurious sequences. In some
embodiments, these constant sequences are selected to promote
incorporation of biotinylated deoxyribonucleotides (e.g.,
biotin-dCTP) into the ends of fragmented molecules during
end-repair.
[0209] In some embodiments, size selection is used to enrich the
library for long fragments to compensate for the diminished
circularization efficiency of long fragments. In some embodiments,
length-dependent binding to SPRI beads is used for size selection.
In some embodiments, agarose or polyacrylamide electrophoresis gel
purification is used for size selection.
[0210] In some embodiments, complete or partial sequencing primer
sequences are included adjacent to the random barcode sequence in
the barcode adapter. This sequence can anneal in downstream PCR to
an oligonucleotide that adds the full sequencing primer sequence.
In some embodiments, sequences corresponding to standard
manufacturer-supplied sequencing primer mixtures are incorporated
to maintain compatibility with such standard primer mixtures. In
some embodiments, custom sequences are used, with a corresponding
custom sequence primer in place of the standard sequencing primer
mixture. Including the eventual sequencing primer sequence proximal
to the barcode in the adapter can have at least two benefits:
[0211] (a) Because the sequencing read begins with the sequence
directly downstream of the sequencing primer sequence, the barcode
sequence is located at the beginning of one of the two paired-end
sequencing reads. After the barcode sequence, the read continues
directly into unknown region derived from the middle of the target
molecule. This method can ensure that the random barcode is easily
identified, and can avoid wasting sequencing capacity by repeatedly
sequencing the region on the upstream side of the barcode (which
derives from the same end of the original target molecule).
[0212] (b) The presence of a primer sequence adjacent to the
barcode sequence can provide a simple way to distinguish DNA
fragments containing barcodes from fragments that do not contain
barcodes. These latter fragments can arise when a copy of the
amplified target molecule is broken more than once, creating two
end fragments with barcode sequences and one or more middle
fragments without barcodes. Sequencing these barcode-free fragments
wastes sequencing capacity because they contain no barcode sequence
to link them to a parent DNA molecule.
[0213] In some embodiments, following fragmentation,
circularization, and shearing, an asymmetric adapter is ligated to
both ends the fragment. Is some embodiments, this adapter is
composed of two oligonucleotides, one of which is longer than the
other. In some embodiments, the shorter oligonucleotide is
complimentary to a portion of the longer oligonucleotide, and upon
annealing creates a ligation-competent adapter with a 3' dT-tail
suitable for specific ligation to the dA-tailed fragment. In some
embodiments, annealing creates a ligation-competent adapter with a
3' dA-tail suitable for specific ligation to the dT-tailed
fragment. In some embodiments, annealing creates a
ligation-competent adapter with a blunt end suitable for ligation
to a blunt-ended fragment. In some embodiments, the adapter
sequence is complimentary to a PCR primer that adds the second
sequencing primer sequence by overlap-extension PCR, but only the
longer of the two oligonucleotides is long enough to productively
anneal to this primer during PCR. As a result, each of the two
strands of the fragment can have an annealing-competent sequence at
exactly one end. The second PCR primer in the reaction can anneal
to the partial sequence adjacent to the barcode. As a result of
this aspect, the desired fragment is in some cases the only
exponentially amplified PCR product (e.g., which begins with a
sequence complementary to at least part of the first sequencing
primer, is followed by the barcode sequence and unknown sequence
from the center of the target molecule, and ends with a sequence
complementary to at least part of the second sequencing
primer).
[0214] The method can be used to sequence the genome of an organism
(e.g., an organism having multiple copies of each chromosome),
single cell or virus haplotyping (e.g., B-cells, cancer stem cells,
virus evolution), RNA sequencing (e.g., splice variants at
multi-exon junctions, short sequence reads matching multiple sites
in the genome), sequencing microbial populations (e.g., microbiome
including pathogenecity islands, environmental microbiology
including enzyme pathways like PKS or NRPS, sequencing of 16S rRNA
including V4 region or full sequence.
Methods for Linking Genotype to Phenotype
[0215] Biopolymers such as proteins and nucleic acids can fold into
three-dimensional structures and perform a diverse set of
functions. In nature, these molecules perform a range of valuable
functions: they efficiently catalyze chemical reactions,
selectively bind desired target molecules, serve as mechanical
scaffolds, assemble into materials, etc. A number of methods have
been developed for the adaptation of natural biomolecules to
perform tasks of interest to humans. Such tasks include catalyzing
industrially important reactions or binding to medically relevant
targets in the body. Evolutionary methods have been extensively
used to modify natural biomolecules. These techniques use largely
random methods to generate collections ("libraries") of variants,
which are tested for the desired properties. Rational,
computational, and intuitive methods are also used to design new
molecules, modify natural molecules, or inform library creation.
Methods for screening variants for desired properties generally
fall into one of two classes. In the first class, a small enough
number of variants is tested that each gene can be synthesized
specifically, and each can be tested within a location (for
example, a test tube or a microtiter plate well) that is known to
contain that specific sequence. This type of experiment links
information from any desired set of phenotypic assays with sequence
information for each variant, but it is limited to a relatively
small number of variants. In the second class, a larger number of
variants are tested, but only a subset is selected for sequencing
(nucleic acids are sequenced directly, while in the case of
proteins the encoding nucleic acid is sequenced). The variant genes
in this case are generally synthesized combinatorially, and their
individual sequences are not known until they are determined by
sequencing reactions. As before, this type of approach provides
linked sequence-activity data for only a relatively small number of
variants.
[0216] When multiple improved variants are found, it is often
desirable to combine the causative mutations into a single variant,
since the effects of beneficial mutations are often additive or
compounding. Statistical methods are increasingly being
incorporated into these approaches to help improve the search
efficiency in the face of overwhelming combinatorial complexity. By
sequencing a number of mutant genes and measuring the activity of
the proteins they encode, the effects of individual mutations can
be statistically isolated, and the best mutations can be identified
more quickly. However, the need to either individually synthesize
or individually sequence interesting variants drastically limits
the amount of information that can be collected. Recently, "deep"
sequencing has been used to simultaneously sequence thousands of
mutants that survive a functional selection. This technique allows
unprecedented statistical power. However, it is limited to binding
proteins and enzymes with activity amenable to selections (for
example, bond-forming enzymes or those whose activity can be linked
to cell survival or growth). In addition, the prevalence of a
mutant within the selected population is the only indication of its
activity relative to other mutants.
[0217] In one aspect, the present disclosure fulfills a need for
generation of large numbers of linked molecular genotype/phenotype
pairs. In some embodiments, the genotype/phenotype pairs can be
analyzed using statistical methods and can be optionally used to
create biological molecules having superior and/or new
properties.
[0218] In one aspect, the present disclosure fulfills a need for
generation of large numbers of linked molecular genotype/phenotype
pairs. In some embodiments, the genotype/phenotype pairs can be
analyzed using statistical methods and can be optionally used to
create biological molecules having superior and/or new
properties.
[0219] In one embodiment, the sequences of nucleic acids are
associated with positions on an array, and the phenotypes of the
encoded variant molecules are determined in parallel at those
positions. In some embodiments, measurements of the properties of
interest of each variant are collected and linked to information
allowing the identification, reproduction, or analysis of the
sequence of each variant. This method can be applied to many types
of biomolecular function, and it may provide a direct link between
sequence information and one or more specific phenotypic
characteristics. The methods described herein produce linked
sequence-phenotype data for a large number of variants.
[0220] In one embodiment, the variant molecules are proteins or
peptides. In other embodiment, the variant molecules are nucleic
acids, small molecules encoded by nucleic acids, proteins or
peptides containing non-natural amino acids, or non-protein
foldamers, such as peptoids or beta-peptides, encoded by nucleic
acids.
[0221] Next-generation sequencing machines use massively parallel
arrays to sequence millions of DNA molecules simultaneously. The
present method includes modification of these, or similar machines
to measure enzyme activity at the same array position at which is
sequenced all or part of the encoding gene, or a short barcode
sequence that can be connected to the full gene sequence. An
emulsion-based method can be used to attach an enzyme and its
encoding DNA to the same microbead. Each enzyme can then be assayed
for activity at the same position at which sequencing data that
directly or indirectly identifies the genotype is collected.
Statistical analysis of the millions of linked sequence/activity
data points can then inform subsequent rounds of designs.
[0222] Read length limitations currently prevent more than a small
stretch of sequence from being determined at once, but read lengths
continue to increase, and within a few years sequencing of entire
genes in a single read may be possible. For example, each position
on the array can contain a nanopore-based sensor, which can detect
enzymatic products as they pass through or occlude the pore, and
also sequence the encoding DNA.
[0223] Alternatively, a sequence outside the coding region can be
sequenced on the array. This region can be short enough to simplify
and facilitate sequencing, yet long enough to serve as a unique
identifier of the corresponding full-length gene sequence. Because
this short barcode sequence can be determined on the array, at the
same position as phenotypic data collection, the barcode can serve
to link the array address of a particular variant with genetic
information that can be used to track the variant after it is
removed from its position on the array. The short barcode region
can be amplified by emulsion PCR upstream to produce sufficient
copies for sequencing. For example, these copies can be attached to
the surface of the same microbead as the full gene and the protein
product. The small size of this amplicon can be conducive for
efficient amplification in emulsion PCR. The full gene can also be
amplified in the same or a separate emulsion PCR as needed to
increase protein expression. The barcode sequence can be completely
degenerate (i.e., poly-N) or the degeneracy can be constrained to
facilitate sequence determination. For example, the sequence can
comprise positions allowed to be A or T alternating with positions
allowed to be G or C, which can reduce or eliminate potential
problems experienced by some sequencing methods when sequencing
homopolymer runs. The degenerate region can also be flanked or
interspersed with partially or fully defined positions to assist
with quality control in downstream computational analysis.
[0224] Given a suitable long-read technology, the present
disclosure includes sequencing a short barcode region on the array,
collecting the variant genes off the array, amplifying and/or
manipulating the DNA as needed to prepare it for long-read
sequencing, and then sequencing the full-length genes with a
long-read method to generate a single sequence that spans the
barcode sequence and the full gene sequence. The full gene sequence
can be thereby linked to the corresponding phenotypic information
collected on the array by virtue of the barcode sequence, which is
linked to the array position by sequencing on the array, and linked
to the full gene sequence by a long read.
[0225] Sequencing can be based on measuring fluorescence or pH.
Fluorescence is commonly used to measure enzymatic activity, as
fluorogenic substrates can be created for many enzymatic activities
of interest. Described herein is use of fluorescence-based machines
to measure the activity of an enzyme and collect information that
directly or indirectly determines the sequence of its co-localized
encoding gene. Examples of cyclic array sequencing by ligation or
by pyrosequencing include Shendure, J., Porreca, G. J., Reppas, N.
B., Lin, X., McCutcheon, J. P., Rosenbaum, A. M., Church, G. M.
(2005). Accurate multiplex polony sequencing of an evolved
bacterial genome. Science (New York, N.Y), 309(5741), 1728-32.
doi:10.1126/science.1117389, which is hereby incorporated in its
entirety, and Margulies, M., Egholm, M., Altman, W. E., Attiya, S.,
Bader, J. S., Bemben, L. a, Rothberg, J. M. (2005). Genome
sequencing in microfabricated high-density picolitre reactors.
Nature, 437(7057), 376-80. doi:10.1038/nature03959, each of which
is hereby incorporated in its entirety.
[0226] For example, the Ion Torrent PGM calls bases by detecting
the minute change in pH caused by the protons released when DNA
polymerase incorporates a new base (Rothberg, J. M., Hinz, W.,
Rearick, T. M., Schultz, J., Mileski, W., Davey, M., . . .
Bustillo, J. (2011). An integrated semiconductor device enabling
non-optical genome sequencing. Nature, 475(7356), 348-52.
doi:10.1038/nature10242, which is hereby incorporated in its
entirety).
[0227] However, many other interesting reactions also cause pH
changes. As described herein, an apparatus containing such chips
may be used to provide massively parallel activity measurements and
sequences of enzymes that catalyze any reaction involving the
release or uptake of ions. The present method of collecting coupled
activity and sequence data from enzymes with a wide range of
activities rapidly accelerates understanding of enzyme function and
the engineering of enzymes with novel activities.
[0228] Described herein are methods to co-locate nucleic acids and
their encoded proteins on an array, such that an apparatus capable
of the parallel measurement of one or more signals (e.g., via one
or more sensors) such as fluorescence, luminescence, temperature
change, or pH change can record both the sequence of all or part of
the nucleic acid or a short barcode nucleic acid uniquely
associated with the full nucleic acid, and the phenotype of the
corresponding protein. In some cases, the one or more signals are
proportional to a phenotype or relatable to a phenotype by a
calibration curve. Sequence data and one or more types of
phenotypic data may be collected in separate reactions, but they
are linked by virtue of occurring at the same (or otherwise
connected or related) physical locations on the array.
[0229] The methods may similarly be used to collect linked genotype
and phenotype information from nucleic acid aptamers, proteins
containing non-canonical amino acids, small molecules encoded by
nucleic acids, proteins or peptides alone using protein sequencing
methods, and so on.
[0230] In one embodiment, DNA molecules are attached to microbeads
or any suitable solid support. Attachment can be achieved by any
suitable method, including binding of a biotin or double-biotin
group attached to the DNA to streptavidin or avidin proteins
attached to the surface of the microbeads. This may result in each
bead binding roughly one DNA molecule. The beads may also be
incubated with biotinylated primers for use in the following
emulsion PCR. The beads are then suspended in a solution containing
PCR reagents, which is emulsified into a continuous oil phase. All
or a portion of the DNA is then amplified by emulsion PCR, and some
fraction of the synthesized DNA copies are attached to the bead.
The emulsion is broken and the beads are pooled and washed. The
beads are now ready for sequencing by technologies including the
Ion Torrent, Roche/454, or Life Technologies APG systems. At this
point, in one embodiment, the beads are incubated with biotinylated
antibodies specific for a peptide tag. They are then washed and
suspended in a solution containing the required components for
cell-free protein synthesis and again emulsified into an immiscible
phase. Within the emulsion droplets, the clonal DNA is transcribed
to produce mRNA, which is translated to produce the encoded variant
protein. The protein is fused to the peptide tag for which the
bead-bound antibodies are specific, such that the produced protein
becomes physically linked to the same bead to which is also linked
its encoding DNA. The production of such microbead-DNA-protein
complexes has been described in the literature (Stapleton J A,
Swartz J R. Development of an In Vitro Compartmentalization Screen
for High-Throughput Directed Evolution of [FeFe] Hydrogenases PLoS
ONE. 2010; 5(12):e10554, which is hereby incorporated in its
entirety.)
[0231] In one embodiment, the beads are then applied to an array
and analyzed with an apparatus capable of (i) sequencing bead-bound
DNA in parallel using technology such as that used in Ion Torrent,
Roche/454, or Life Technologies APG systems, and (ii) delivering
solutions to create the conditions for a desired protein assay,
other than those used in the sequencing reaction, and measuring in
parallel position-linked signals (e.g., fluorescence, luminescence,
temperature change, or pH change) that correspond to the
performance of each protein variant in the assay. Application of
the parallel sequencing technology provides sequence information
associated with each position on the array. All or part of the DNA
can be sequenced, in one step or in multiple steps (e.g., each with
different priming oligonucleotides). Prior or subsequent to
sequencing, application of the parallel assay technology provides
one or more measurements of the phenotype of the protein in one or
more assays, again associated with each position on the array.
Linked genotype-phenotype information can be generated for a large
number of variants in parallel.
[0232] For example, the green fluorescent protein (GFP) is widely
used as an in vivo marker in biological studies, and has been the
target of much protein engineering to understand its function and
to generate variants with improved properties such as stability,
maturation speed, and altered spectral properties. The methods
described herein may be used to rapidly gather a large amount of
sequence-activity data for use in GFP engineering. A library of
biotinylated genes encoding GFP variants tagged with unique barcode
sequences may be generated, for example, by error-prone PCR with a
degenerate barcode region designed into one of the primers. The
genes are attached to microbeads and amplified by emulsion PCR. The
barcode region alone can be separately amplified by emulsion PCR,
such that many copies of the barcode sequence are attached to the
microbead. The genes can be transcribed and translated by emulsion
cell-free protein synthesis as described previously. The
microbeads, which display clonal variant DNA and its encoded
variant GFP protein, are applied to an array. The barcode DNA on
each bead is sequenced in parallel using known next-generation
sequencing technology. Following (or prior to) the sequencing
stage, the GFP variant proteins attached to each bead are assayed.
In one example, the array is exposed to light whose wavelength is
controlled by one or more filters, and the machine measures the
fluorescent light emitted from each position on the array that
passes through a second set of one or more filters. Multiple
measurements may be performed sequentially, changing the input and
output filters with each measurement to acquire detailed
information on the fluorescence properties of each variant. The
temperature and chemical environment (e.g., the concentration of
guanidinium hydrochloride or urea) may also be varied or titrated
while measuring the fluorescent output of each variant, providing
information on additional properties of the variants (e.g.,
stability). If a superior variant were present on the array, the
linked sequence information collected in sequencing may be used to
reproduce that protein for further characterization. Alternatively,
the large number of linked sequence/phenotype measurements may be
analyzed statistically to identify mutations or combinations
thereof that are beneficial for GFP performance, and these
mutations can be recombined in one or a few designed variants or in
a new library for further rounds of screening. In one embodiment, a
machine-learning algorithm is trained to predict the properties of
a GFP variant of arbitrary sequence. The large datasets provided by
the methods described herein may be useful in the engineering of
new proteins and in furthering scientific understanding of how
proteins and enzymes fold and function.
[0233] In some instances, emulsion PCR is less efficient with
longer DNA templates. In some embodiments multiple sets of primers
may be used in emulsion PCR, simultaneously or sequentially, to
amplify shorter stretches of the DNA sequence. These short
sequences lack an RNA polymerase promoter and are not transcribed
in cell-free protein synthesis, but are suitable for sequencing.
The entire gene can be represented in a set of such short
amplicons, which can be sequenced sequentially on the array using
different priming oligonucleotides. This embodiment may include
emulsion PCR to amplify the entire gene if such amplification is
necessary to eventually synthesize enough protein for the desired
phenotypic assays.
[0234] Many other similar embodiments may be imagined by those
familiar with the art. For example, emulsion PCR could be omitted,
or replaced with in vitro transcription, followed in some
embodiments by a reverse-transcription. Alternatively, biotinylated
RNA could be transcribed in bulk solution and then attached to
microbeads.
[0235] While the above descriptions have focused on the binding of
molecules to microbeads, the method is not limited in this regard.
Nucleic acids can be bound directly to surfaces such as glass. The
encoded proteins can be synthesized prior to or following nucleic
acid binding to the chip and bound to the same surface or to the
nucleic acids themselves (e.g., by ribosome display, RNA display,
or DNA display). Surface-bound nucleic acids can optionally be
amplified before or after transcription or translation by methods
including bridge PCR. Binding the nucleic acids to a surface may
allow other high-throughput sequencing technologies to be used,
including those developed by Illumina/Solexa and Helicos
BioSciences. Alternatively, single nucleic acid/protein complexes
such as those that result from ribosome display, RNA display, or
DNA display can be sequenced by technologies such as those
developed by Pacific Biosciences, or by nanopore sequencing.
[0236] In another embodiment, the active molecule is RNA rather
than protein. In this case, a number of approaches can be used,
including but not limited to the following:
[0237] (i) a protocol similar to the microbead-attachment protocol
described above can be used, but the cell-free protein synthesis is
replaced by in vitro transcription within the emulsion. The
phenotypes of the resulting RNAs are measured as described above
(e.g., pH changes).
[0238] (ii) a microbead-attachment protocol can be used, wherein
the DNA and the microbead are co-compartmentalized during an in
vitro transcription that results in decoration of the microbead
with RNA. The RNA is then sequenced directly or reverse-transcribed
to generate DNA for sequencing.
[0239] (iii) single molecules of RNA are attached to beads,
surfaces, or surface-bound molecules such as polymerases, and
sequenced directly or reverse-transcribed to generate DNA for
sequencing, prior to or following single-molecule
characterization.
[0240] In some embodiments, for example where enzymatic rates are
of interest, methods are described herein for estimating
approximately how many copies of the enzyme were bound to a given
microbead during protein synthesis. This can be accomplished in a
number of ways. For example, the enzyme can be linked at a defined
stoichiometry to a molecule or fusion of known characteristics.
Measurement of a signal from the array position specific to this
calibration molecule allows determination of the number of copies
of the molecule of interest at each position in the array. For
example, the number of these control molecules can be determined by
measuring fluorescence, luminescence, temperature change, or pH
change as a result of enzymatic activity or binding to a probe
molecule such as an antibody linked to a fluorescent molecule, an
enzyme, or an enzymatic substrate.
[0241] In some embodiments, for example where binding is of
interest, the molecule to be bound is conjugated or fused to an
enzyme capable of generating a signal with a high turnover rate, so
that each bound molecule generates an amplified signal to
facilitate detection. In some embodiments, the substrate and/or
product of this reaction is attached to microbeads or to the array
surface to preserve the localization of the signal within the
particular array position.
[0242] In another embodiment, the nucleic acid sequences to be
tested are spotted or printed directly onto known positions on the
array. This can be done by any one of a number of technologies,
including ink-jet or photolithography-based methods. In some
embodiments, the nucleic acid is RNA, but it can also be DNA, in
which case it may be transcribed by any method that preserves the
spatial information that locates the sequence on the array.
Ligation between the DNA and corresponding RNA is one such
mechanism. Array-bound RNA may be translated using methods such as
ribosome display or RNA display, wherein the newly synthesized
protein remains spatially associated with its encoding RNA or DNA
or the array. Alternatively, peptides or proteins with specific
sequences can be synthesized directly onto defined positions on the
array by solid-phase synthesis. In these embodiments sequencing is
not necessary, as the sequence of the nucleic acid printed in each
location is known. Phenotypic characterization takes place in
parallel on the array as described.
[0243] In another embodiment, oligonucleotides containing "barcode"
sequences, each of which refer to a specific full-length variant
gene, are printed onto an array. Nucleic acid/protein complexes
then attach to the array by way of hybridization between the
nucleic acid and the bound oligonucleotides. The nucleic acids
contain complementary barcode sequences that allow specific
annealing to a particular array-bound oligonucleotide. In some
embodiments, nucleic acid/protein complexes (where the nucleic acid
can be RNA or DNA, and can be complexed with its encoded protein by
ribosome display, RNA display, DNA display, mutual attachment to a
microbead, and so on) are synthesized and assembled in bulk
solution and then directed to known positions on an array. On-array
sequencing is therefore not needed, and long-read sequencing can be
subsequently performed if necessary to link the barcode sequences
with the full-length gene sequences. Parallel, location-linked
phenotypic characterization takes place as described herein. The
protein-associated nucleic acid could contain the open reading
frame along with the barcode, or it could contain only the barcode.
The latter scenario could be accomplished by, for example, binding
a nucleic acid molecule comprising a barcode and an open reading
frame to a microbead, and amplifying only the barcode section by
emulsion PCR such that the bead becomes decorated with many copies
of the barcode sequence. Alternatively, a method similar to DNA
display could be used to attach a barcode sequence directly to the
protein.
[0244] The methods can also be applied in many other areas of
science and engineering. For example, it could be used to rapidly
characterize unknown open reading frames from, e.g., environmental
samples. These genes could be expressed, displayed on the array,
and exposed sequentially to a battery of tests for common enzymatic
activities, binding partners, biophysical properties, and the
like.
[0245] The method may be used to modify the properties of an
existing enzyme or ribozyme by directed evolution. A mutant library
is generated from a starting parent gene. The library is analyzed
using the described method, which provides data describing the
complete or partial sequence and phenotype of each mutant. This
data is used to generate a new mutant library, which can be based
on one or more mutants with desirable properties identified by the
method, or which can be combinatorially assembled from
oligonucleotides containing one or more mutations identified by the
method as being statistically associated with desirable phenotypes.
This process is iteratively repeated for as many cycles as
desired.
[0246] It may be desirable to sequence the nucleic acids more than
once while maintaining their positions on the array, for example to
ensure sequencing accuracy. Many parallel sequencing technologies
have read lengths that are short relative to the length of a
typical gene. In some embodiments, different regions of a nucleic
acid may be sequenced in multiple sequential sequencing runs. These
partial sequences are collected sequentially but remain associated
with the same array position. The partial sequences may then be
combined using overlapping regions or by comparison to a known
parent or reference sequence. The partial sequences may be
generated by sequencing regions of the same nucleic acid molecule.
Alternatively, sections of the long nucleic acid polymer that
contains the open reading frame can be individually amplified to
create a number of smaller nucleic acid molecules, which remain
associated with the parent, e.g. by binding to the same bead
following emulsion PCR. These smaller nucleic acids can then be
sequenced, and these partial sequences combined as described
previously.
[0247] In some aspects, an array described herein comprises at
least about 1, 2, 10.sup.1, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11 or
more sensors. In some aspects, an array described herein comprises
at most about 10.sup.11, 10.sup.10, 10.sup.9, 10.sup.8, 10.sup.7,
10.sup.6, 10.sup.5, 10.sup.4, 10.sup.3, 10.sup.2, 10.sup.1, 2
sensors or 1 sensor. A sensor may measure a signal associated with
a signal associated with fluorescence, pH change, temperature
change, luminescence, or any combination thereof. In some aspects,
an array described herein may be interrogated by a sensor. Such a
sensor may measure a signal associated with a signal associated
with fluorescence, pH change, luminescence, temperature change or
any combination thereof associated with the array. In some aspects,
an array comprises one or more chemical field-effect transistor
(chemFET) sensors.
[0248] In some aspects, a phenotype described herein may be any
phenotype of interest. Non-limiting examples of phenotypes include
enzyme specificity, binding affinity, binding specificity and
stability when exposed to a chemical condition or a temperature. In
some aspects, a method includes contacting proteins to a plurality
of solutions comprising substrates at a plurality of
concentrations. In some aspects, a method includes contacting
proteins to a plurality of solutions comprising ligands at a
plurality of concentrations. In some aspects, a method includes
measuring a phenotype at a plurality of temperatures.
Computer Control Systems
[0249] The present disclosure provides computer control systems
that are programmed to implement methods of the disclosure. FIG. 9
shows a computer system 901 that is programmed or otherwise
configured to operate instrumentation (e.g. a thermal cycler, fluid
handling apparatuses including pumps and valves, a sequencing
instrument, a sequencing platform, etc.), analyze and store
sequencing reads, perform sequence assembly, store results of a
sequence assembly, display data (e.g., results of sequencing
analysis, instrument operational parameters, etc). The computer
system 901 can regulate various aspects of devices (e.g., thermal
cyclers, fluid handling apparatuses including pumps and valves,
sequencing instrumentation, sequencing platforms, etc.), sequence
read analysis methods and sequence assembly methods described
herein. The computer system 901 can be an electronic device of a
user or a computer system that is remotely located with respect to
the electronic device. The electronic device can be a mobile
electronic device.
[0250] The computer system 901 includes a central processing unit
(CPU, also "processor" and "computer processor" herein) 905, which
can be a single core or multi core processor, or a plurality of
processors for parallel processing. The computer system 901 also
includes memory or memory location 910 (e.g., random-access memory,
read-only memory, flash memory), electronic storage unit 915 (e.g.,
hard disk), communication interface 920 (e.g., network adapter) for
communicating with one or more other systems, and peripheral
devices 925, such as cache, other memory, data storage and/or
electronic display adapters. The memory 910, storage unit 915,
interface 920 and peripheral devices 925 are in communication with
the CPU 905 through a communication bus (solid lines), such as a
motherboard. The storage unit 915 can be a data storage unit (or
data repository) for storing data. The computer system 901 can be
operatively coupled to a computer network ("network") 930 with the
aid of the communication interface 920. The network 930 can be the
Internet, an internet and/or extranet, or an intranet and/or
extranet that is in communication with the Internet. The network
930 in some cases is a telecommunication and/or data network. The
network 930 can include one or more computer servers, which can
enable distributed computing, such as cloud computing. The network
930, in some cases with the aid of the computer system 901, can
implement a peer-to-peer network, which may enable devices coupled
to the computer system 901 to behave as a client or a server.
[0251] The CPU 905 can execute a sequence of machine-readable
instructions, which can be embodied in a program or software. The
instructions may be stored in a memory location, such as the memory
910. The instructions can be directed to the CPU 905, which can
subsequently program or otherwise configure the CPU 905 to
implement methods of the present disclosure. Examples of operations
performed by the CPU 905 can include fetch, decode, execute, and
writeback.
[0252] The CPU 905 can be part of a circuit, such as an integrated
circuit. One or more other components of the system 901 can be
included in the circuit. In some cases, the circuit is an
application specific integrated circuit (ASIC).
[0253] The storage unit 915 can store files, such as drivers,
libraries and saved programs. The storage unit 915 can store user
data, e.g., user preferences and user programs. The computer system
901 in some cases can include one or more additional data storage
units that are external to the computer system 901, such as located
on a remote server that is in communication with the computer
system 901 through an intranet or the Internet.
[0254] The computer system 901 can communicate with one or more
remote computer systems through the network 930. For instance, the
computer system 901 can communicate with a remote computer system
of a user. Examples of remote computer systems include personal
computers (e.g., portable PC), slate or tablet PC's (e.g.,
Apple.RTM. iPad, Samsung.RTM. Galaxy Tab), telephones, Smart phones
(e.g., Apple.RTM. iPhone, Android-enabled device, Blackberry.RTM.),
or personal digital assistants. The user can access the computer
system 901 via the network 930.
[0255] Methods as described herein can be implemented by way of
machine (e.g., computer processor) executable code stored on an
electronic storage location of the computer system 901, such as,
for example, on the memory 910 or electronic storage unit 915. The
machine executable or machine readable code can be provided in the
form of software. During use, the code can be executed by the
processor 905. In some cases, the code can be retrieved from the
storage unit 915 and stored on the memory 910 for ready access by
the processor 905. In some situations, the electronic storage unit
915 can be precluded, and machine-executable instructions are
stored on memory 910.
[0256] The code can be pre-compiled and configured for use with a
machine having a processer adapted to execute the code, or can be
compiled during runtime. The code can be supplied in a programming
language that can be selected to enable the code to execute in a
pre-compiled or as-compiled fashion.
[0257] Aspects of the systems and methods provided herein, such as
the computer system 901, can be embodied in programming. Various
aspects of the technology may be thought of as "products" or
"articles of manufacture" typically in the form of machine (or
processor) executable code and/or associated data that is carried
on or embodied in a type of machine readable medium.
Machine-executable code can be stored on an electronic storage
unit, such as memory (e.g., read-only memory, random-access memory,
flash memory) or a hard disk. "Storage" type media can include any
or all of the tangible memory of the computers, processors or the
like, or associated modules thereof, such as various semiconductor
memories, tape drives, disk drives and the like, which may provide
non-transitory storage at any time for the software programming.
All or portions of the software may at times be communicated
through the Internet or various other telecommunication networks.
Such communications, for example, may enable loading of the
software from one computer or processor into another, for example,
from a management server or host computer into the computer
platform of an application server. Thus, another type of media that
may bear the software elements includes optical, electrical and
electromagnetic waves, such as used across physical interfaces
between local devices, through wired and optical landline networks
and over various air-links. The physical elements that carry such
waves, such as wired or wireless links, optical links or the like,
also may be considered as media bearing the software. As used
herein, unless restricted to non-transitory, tangible "storage"
media, terms such as computer or machine "readable medium" refer to
any medium that participates in providing instructions to a
processor for execution.
[0258] Hence, a machine readable medium, such as
computer-executable code, may take many forms, including but not
limited to, a tangible storage medium, a carrier wave medium or
physical transmission medium. Non-volatile storage media include,
for example, optical or magnetic disks, such as any of the storage
devices in any computer(s) or the like, such as may be used to
implement the databases, etc. shown in the drawings. Volatile
storage media include dynamic memory, such as main memory of such a
computer platform. Tangible transmission media include coaxial
cables; copper wire and fiber optics, including the wires that
comprise a bus within a computer system. Carrier-wave transmission
media may take the form of electric or electromagnetic signals, or
acoustic or light waves such as those generated during radio
frequency (RF) and infrared (IR) data communications. Common forms
of computer-readable media therefore include for example: a floppy
disk, a flexible disk, hard disk, magnetic tape, any other magnetic
medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch
cards paper tape, any other physical storage medium with patterns
of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other
memory chip or cartridge, a carrier wave transporting data or
instructions, cables or links transporting such a carrier wave, or
any other medium from which a computer may read programming code
and/or data. Many of these forms of computer readable media may be
involved in carrying one or more sequences of one or more
instructions to a processor for execution.
[0259] The computer system 901 can include or be in communication
with an electronic display 935 that comprises a user interface (UI)
940 for providing, for example, operation parameters of an
instrument (e.g., a thermal cycler, a sequencing instrument, fluid
handling instrumentation), instrument performance, parameters of a
sequence assembly method, results and associated statistics of a
sequence assembly data, etc. Examples of UI's include, without
limitation, a graphical user interface (GUI) and web-based user
interface.
[0260] Methods and systems of the present disclosure can be
implemented by way of one or more algorithms. An algorithm can be
implemented by way of software upon execution by the central
processing unit 905. The algorithm can, for example, initiate
electronic signals that are processed to operate instrumentation
(e.g. a thermal cycler, fluid handling apparatuses including pumps
and valves, a sequencing instrument, a sequencing platform, etc.),
analyze and store sequencing reads, perform sequence assembly
and/or store results, display data (e.g., results of sequencing
analysis, instrument operational parameters, etc) to a user,
transmit to or receive data from a remote computer system, etc.
EXAMPLES
[0261] Additional aspects and details of the invention will be
apparent from the following examples, which are intended to be
illustrative rather than limiting.
Example 1--Standard Protocols
[0262] Standard protocols used in many Examples of the disclosure
were provided. A solution of two oligonucleotides (e.g., where the
first (the barcode-containing oligo) was any of oligo 1, oligo 2,
oligo 3, or oligo 4, and the second (the extension oligo) was any
of oligo 5, oligo 6, or oligo 7, where oligo 5 is used with oligo 1
or oligo 4; oligo 6 is used with oligo 1, oligo 2, or oligo 4; and
oligo 7 is used with oligo 3--the various oligos corresponding to
those shown in Table 1 below), at 2 .mu.M and 5 .mu.M, respectively
in NEBuffer 2 (New England Biolabs (NEB), Ipswich, Mass.) was
heated to 95.degree. C. for 10 minutes and allowed to cool to
37.degree. C. over 30 minutes. Five units of Klenow exo- (NEB) and
0.3 mM each dNTP (NEB) were added and the mixture was incubated at
37.degree. C. for 60 minutes.
[0263] The library DNA to be sequenced was linearized and
fragmented to the desired size by restriction digestion,
fragmentation, or PCR as necessary. In some aspects, depending on
the source of the nucleic acid and the goals of the project, the
nucleic acid is fragmented into sizes from about 1 kb to about 20
kb. For example, genomic DNA is usually sheared to about 10 kb. In
other aspects, genes of about 3 kb are the sequence of interest.
The gene can be amplified from source DNA or cut out of a larger
genome with restriction enzymes. The DNA to be sequenced was
typically diluted to 50 .mu.L at 10 ng/.mu.L and fragmented into
.about.10 kb pieces with a g-TUBE (Covaris, Woburn, Mass.) by
centrifugation at 4200 g according to the manufacturer's
protocol.
[0264] The DNA was end-repaired with the NEBNext End Repair Module
(NEB) according to the manufacturer's suggested protocol and
purified with a Zymo DNA Clean & Concentrator column (Zymo
Research, Irvine, Calif.) and eluted in 20 .mu.L of buffer EB (an
elution buffer used in eluting DNA). The DNA was then dT-tailed by
incubation in 1.times. NEB buffer 2 with 1 mM dTTP (Life
Technologies, Grand Island, N.Y.), 5 units Klenow exo-, and 10
units polynucleotide kinase at 37.degree. C. for 1 hour.
[0265] 250 fmol of library DNA and 5 pmol of barcoded tipartite
adapters comprising an outer PCR primer region, an inner sequencing
primer region, and a central barcode region were ligated with
TA/Blunt MasterMix (NEB) according to the manufacturer's protocol,
purified with a Zymo column or with gel purification with size
selection with the Qiagen Gel Extraction kit and eluted in 20 .mu.L
of buffer EB. The tripartite adapters, see, e.g., oligo 1 in Table
1, are designed so that barcode number takes into consideration
target number. For example, an adapter comprising a 16N barcode
works for about 10 to about 20 million target sequences.
[0266] Two single-stranded oligonucleotides are ordered from a
supplier, annealed together, and the shorter one is extended to
form the double-stranded adapter. The number of possible barcode
sequences is 4.sup.11, where n is the number of degenerate bases.
That number should be at least 100 times higher than the number of
DNA molecules to be tagged to ensure that each molecule receives
two unique tags. For example, n=16 has been used in experiments
described herein. 4.sup.16=4.3 billion. In various aspects, the
barcode is made shorter (to maximize the portion of the sequencing
read that reads target sequence) or longer (to ensure that no two
molecules get identical barcodes).
[0267] Oligo 5, oligo 6 and oligo 7, shown in Table 1 below,
represent both the shorter adapter extension oligo described herein
above and the PCR primer (see Rungpragayphan et al., J. Mol. Biol.
318:395-405, 2002). Theoretically, the extension oligo may be any
sequence long enough for primer annealing during PCR. The extension
oligo anneals to the barcode-containing oligo and is extended by
Klenow exo.sup.- polymerase, copying the barcode and forming a
dA-tailed double-stranded adapter. The region on the 5' end of the
barcode-containing oligo is the sequence from the Illumina
Universal sequencing primer. If a different sequencing primer is
used for sequencing, the barcode-containing oligo should be
modified accordingly.
[0268] The adapters were ligated at both ends of the DNA. A single
adapter is ligated to each end of the nucleic acid by including an
overhang on the 3' strand of the non-ligating end, thus blocking
concatamerization on the end of the adapter. Library molecules that
failed to ligate to an adapter at both ends were removed by
incubation with 10 units of exonuclease III (NEB) and 20 units of
exonuclease I (NEB) in NEBuffer 1 for 45 minutes at 37.degree. C.,
followed by 20 minutes at 80.degree. C.
[0269] Oligo 2, shown in Table 1 below, comprises an example of one
strand of the tripartite adapter. The oligo, from 5' to 3',
comprises: (1) NNN, which is an optional degenerate 5' end to
reduce sequence bias of ligation, (2) CCTACACGACGCTCTTCCGATCT,
which is the annealing sequence for oligo 11 (shown in Table 1
below), which adds the Illumina TruSeq Universal adapter during the
final limited-cycle PCR; (3), which is the degenerate barcode
sequence; (4) CC, which is a short defined sequence to confirm that
the previous bases comprise the barcode and to promote biotin-dCTP
incorporation during end repair; (5) AGGAATAGTTATGTGCATTAATGAATGG,
which is an annealing sequence for oligo 6 (shown in Table 1
below), which both extends oligo 2 (shown in Table 1 below) to form
the double-stranded tripartite adapter and is the primer for the
first PCR; and (6) CGCC, which is a short overhanging sequence to
prevent ligation on this end of the tripartite adapter, and which
can be extended to include a primer annealing site for linear
amplification.
[0270] The ligation product was quantified with the Quant-It kit
(Life Technologies) and diluted to about 10,000 molecules per .mu.L
to impose a complexity bottleneck. A complexity bottleneck sets the
number of molecules that are amplified, matching the sequencing
capacity to ensure that each molecule accumulates enough sequencing
reads to assemble long synthetic reads. In this example, ten
thousand molecules of adapter-ligated DNA were amplified by PCR
using a PfuCx polymerase (Agilent Technologies, Santa Clara,
Calif.) or LongAmp Taq DNA polymerase (NEB) and a single primer
(e.g., oligo 6 shown in Table 1 below) at 0.5 mM. The following
thermocycling conditions were carried out: 92.degree. C. for 2
minutes, followed by 40 cycles of 92.degree. C. for 20 seconds,
55.degree. C. for 20 seconds, and 68.degree. C. for 3 minutes/kb,
and followed by a final hold at 68.degree. C. for 10 minutes.
[0271] The PCR products were purified with a Zymo column or a
Qiagen Gel Extraction kit and eluted in 50 .mu.L of buffer EB.
Between 200 ng and one .mu.g of DNA was mixed with 1 unit of USER
enzyme in a 45 .mu.L reaction volume and incubated for 30 minutes
at 37.degree. C. Two .mu.L of 1:5 diluted dsDNA fragmentase (NEB),
100 .mu.g/mL bovine serum albumin, and 5 .mu.L of dsDNA fragmentase
buffer were added and the mixture incubated on ice for 5 minutes.
0.5-2 .mu.L of dsDNA fragmentase (NEB) (volume adjusted based on
amount and length of DNA to be fragmented) were then added and the
mixture incubated at 37.degree. C. for 15 minutes. The reaction was
stopped by addition of 5 .mu.L of 0.5 M EDTA and fragmentation was
confirmed by the presence of a smear on an agarose gel. The DNA was
purified with a Zymo column or 0.8 volumes of Ampure XP beads
(Beckman Coulter, Brea, Calif.), and eluted in 20 .mu.L of buffer
EB.
[0272] Two .mu.L of 10.times. NEBuffer 2 were added and fragmented
DNA was incubated with 0.5 .mu.L of "E. coli DNA ligase for
fragmentase" (NEB) for 20 minutes at 20.degree. C. Three units of
T4 DNA polymerase (NEB), 5 units of Klenow fragment (NEB), and 50
.mu.M of biotin-dCTP (Life Technologies) were added; and the
reaction was incubated for 10 minutes at 20.degree. C. Fifty .mu.M
dGTP, dTTP, and dATP were added and the mixture was incubated for
an additional 15 minutes, purified with a Zymo column or 1 volume
of Ampure XP beads, eluted in 20 .mu.L of buffer EB, and quantified
by absorbance at 260 nm.
[0273] 200-1000 ng of DNA at a final concentration of 1 ng/.mu.L
were mixed with 3000 units of T4 DNA ligase and T4 DNA ligase
buffer to 1.times., and incubated at 16.degree. C. for 16 hours.
Linear DNA was digested by the addition of 10 units of T5
exonuclease and incubation at 37.degree. C. for 60 minutes.
Circularized DNA was purified with a Zymo column and eluted in 130
.mu.L of buffer EB. The DNA was fragmented with an S2 disruptor
(Covaris, Inc., Woburn, Mass.) to lengths of about 500 bp to about
800 bp.
[0274] Twenty .mu.L of Dynabeads M-280 Streptavidin Magnetic Beads
(Life Technologies) were washed twice with 200 .mu.L of 2.times.
B&W buffer (1.times. B&W buffer: 5 mM Tris-HCl (pH 7.5),
0.5 mM EDTA, 1 M NaCl) and resuspended in 100 .mu.L of 2.times.
B&W buffer. The DNA solution was mixed with this bead solution
and incubated for 15 minutes at 20.degree. C. The beads were washed
three times with 200 .mu.L of 1.times. B&W buffer, and twice in
200 .mu.L of buffer EB. At this point, 15% (30 .mu.L) of the beads
were removed to a new tube for two-tube barcode pairing (see
below). The remaining beads were resuspended in NEBNext End Repair
Module solution (New England BioLabs Inc., Ipswich, Mass.) (42
.mu.L water, 5 .mu.L End Repair Buffer, and 2.5 .mu.L End Repair
Enzyme Mix), incubated at 20.degree. C. for 30 minutes, washed
three times with 200 .mu.L of 1.times. B&W buffer, and then
twice with 200 .mu.L of buffer EB. The beads were resuspended in
NEBNext A-tailing Module solution (NEB), incubated at 37.degree. C.
for 30 minutes, and washed three times with 200 .mu.L of 1.times.
B&W buffer, and then twice with 200 .mu.L of buffer EB.
[0275] A 15 .mu.M equimolar mixture of two oligonucleotides (e.g.,
oligos 8 and 9, as set out in Table 1 below) in 1.times. T4 DNA
ligase buffer was incubated at 95.degree. C. for 10 minutes and
allowed to slowly cool to room temperature. The beads were
resuspended in a solution comprising 5 .mu.L of NEB Blunt/TA ligase
master mix (NEB), 0.3 .mu.L of 15 .mu.M adapter oligo solution, and
4 .mu.L of water. The mixture was incubated for 15 minutes at room
temperature. The beads were washed three times with 200 .mu.L of
1.times. B&W buffer, and twice with 200 .mu.L of buffer EB. The
beads were resuspended in a 50 .mu.L PCR solution comprising 36
.mu.L of water, 10 .mu.L of 5.times. Phusion HF DNA polymerase
buffer, 1.25 .mu.L of each of 10 .mu.M solutions of the standard
Illumina Index and Universal primers (oligos 5 and 6 (set out below
in Table 1), and 0.02 units/.mu.L Phusion DNA polymerase (Thermo
Fisher Scientific, Inc., Skokie, Ill.). The following thermocycling
program was used: 98.degree. C. for 30 seconds, followed by 18
cycles of 98.degree. C. for 10 seconds, 60.degree. C. for 30
seconds, and 72.degree. C. for 30 seconds, and a final hold at
72.degree. C. for 5 minutes. The supernatant was retained and the
beads discarded.
[0276] The PCR product was purified with 0.7 volumes of Ampure XP
beads and eluted in 10 .mu.L buffer EB, or 500-900 bp fragments
were size-selected on an agarose gel, gel-purified with the
MinElute Gel Extraction kit, and eluted in 15 .mu.L of buffer EB.
The size distribution of the DNA was measured with an Agilent
bioanalyzer and cluster-forming DNA was quantified by qPCR. The DNA
fragments were sequenced on a MiSeq, NextSeq or HiSeq sequencer
(Illumina) with standard Illumina primers. Illumina provides primer
mixes with their sequencing reagent kits that include sequencing
primers compatible with all of their various sequencing preparation
kits. For example, multiple sequencing kits, each with their own
sequences, are available, and the primer mixture contains primers
compatible with all of the kits. Oligos 8 and 9, set out in Table 1
below, anneal to one another to form the asymmetric adapter. Oligos
10 and 11, set out in Table 1 below, are PCR primers that add the
complete Illumina flowcell sequences. Sequences used in oligo 2,
10, and 11, as set out in Table 1 below, are from the Illumina
Small RNA Kit. One oligo anneals to the asymmetric adapter, while
the other oligo anneals to a region of the barcode adapter that is
now on the interior of the fragment.
[0277] The Illumina sequences were taken from Illumina to ensure
compatibility with the standard sequencing primer mix, but these
sequences can be made longer or shorter or replaced entirely if
corresponding custom sequencing primers are used. In this Example,
16-base random barcodes were used, but any length is adaptable for
use. Additionally, the sequences can be less than completely
degenerate (e.g., allowing only 1, 2, or 3 nucleotides at some or
all positions). In the sequences used in this Example, there was a
2-base constant region outside the barcodes. However, this region
of bases can be modified, i.e., made shorter or longer, or omitted
altogether.
[0278] Moreover, two separate protocols were developed for barcode
pairing, a two-tube protocol and a one-tube protocol. The one-tube
protocol has the advantage of sample preparation occurring entirely
in a single tube. A mixture of two or more barcode-containing
adapters is ligated to the dT-tailed target fragments (e.g., a
mixture of oligo 1 and oligo 2 as shown in Table 1). The adapters
differ in their sequencing primer region. Sequences were derived
from the Illumina Universal and Index primer sequences,
respectively. As a result, approximately half of the target
fragments will have different sequencing regions in the adapters
that ligate to the two ends. Following PCR, some fraction of the
full-length copies will avoid fragmentation, and circularization
will bring the two barcodes together. Downstream limited-cycle PCR
(lcPCR) will fail to amplify molecules that have the same adapter
at each end because the identical sequencing regions outside the
barcode regions will form a tight hairpin upon becoming single
stranded. However, in molecules with different adapters at the
ends, no hairpin will form, and addition of a primer complementary
to the second sequencing region enables amplification of the paired
barcodes. In the computational pipeline, paired-barcode reads are
identified, trimmed of adapter sequences, and parsed to extract the
barcode pairs.
[0279] The two-tube protocol adds the complexity of splitting the
library preparation into two tubes for the last third of the
protocol, one tube to generate barcoded target reads and a second
solely to generate paired barcode reads. The advantage is improved
control of the fraction of the eventual short reads of each type.
In this protocol, only one adapter sequence is used, so all target
molecules ligate the same adapter at both ends. As a result, all
molecules derived from circularized full-length amplicons will form
a tight hairpin during lcPCR, and no paired-barcode reads will be
present in the main sequencing sample. Following attachment to
streptavidin-coated beads and prior to ligation of asymmetric
adapters, a fraction (.about.15%) of the beads are moved to a
second tube. SapI digestion cuts a site in the sequencing region
(taken from the Illumina Multiplexing Sample Prep Oligo Only Kit),
leaving sticky ends. Y-shaped adapters are ligated to the sticky
ends to provide PCR annealing regions, and subsequent lcPCR adds
the requisite sequencing adapter regions and a multiplexing index
that allows barcode-pairing reads to be identified during
analysis.
[0280] Two-tube barcode pairing: Bead-bound DNA was digested with
10 units of SapI in 1.times. CutSmart buffer in a 20 .mu.L total
volume for 1 h at 37.degree. C. The beads were washed three times
with 200 .mu.L of 1.times. B&W buffer and twice with 200 .mu.L
of buffer EB. A 15 .mu.M equimolar mixture of two oligonucleotides
(oligos 12 and 13, as set out in Table 1 below) in 1.times. T4 DNA
ligase buffer was incubated at 95.degree. C. for 2 minutes and
allowed to cool to room temperature over 30 minutes. The beads were
resuspended in a solution comprising 5 .mu.L of NEB Blunt/TA ligase
master mix, 0.5 .mu.L of 15 .mu.M adapter oligo solution, and 4
.mu.L of water. The mixture was incubated for 15 minutes at
4.degree. C. and 15 minutes at 20.degree. C. The beads were washed
twice with 200 .mu.L of 1.times. B&W buffer and twice with 200
.mu.L of buffer EB. For amplification by limited-cycle PCR, the
beads were resuspended in a 50 .mu.L PCR solution comprising 36
.mu.L of water, 10 .mu.L of 5.times. Phusion HF DNA polymerase
buffer, 1.25 .mu.L of each of 10 .mu.M solutions of two primers
(oligos 11 and 14, as set out in Table 1 below, with oligo 14 (as
shown in Table 1) selected to have a different multiplexing index
than oligo 10 (as shown in Table 1) used above), and 0.02
units/.mu.L Phusion DNA polymerase (Thermo Fisher Scientific). The
following thermocycling program was used: 98.degree. C. for 30
seconds, followed by 18 cycles of 98.degree. C. for 10 seconds,
60.degree. C. for 30 seconds, and 72.degree. C. for 30 seconds, and
a final hold at 72.degree. C. for 5 minutes. The supernatant was
retained and the beads discarded. DNA was purified with 1.8 volumes
of Ampure XP beads and eluted in 10 .mu.L buffer EB. The expected
product size of .about.170 bp was confirmed by agarose gel
electrophoresis and Agilent bioanalyzer. Cluster-forming DNA was
quantified by qPCR. The DNA fragments were mixed with the main
library so as to comprise 1-5% of the total molecules, and
sequenced on an Illumina MiSeq, NextSeq, or HiSeq with standard
Illumina primer mixtures.
[0281] Single-tube barcode pairing: Oligos 1 and 2 (as shown in
Table 1) were mixed, extended with oligo 6 (as shown in Table 1),
and ligated to dT-tailed target fragments as above. The library
preparation protocol was carried out as above, except that no extra
barcode-pairing was completed. Limited-cycle PCR was performed with
1.25 .mu.L of a 10 micromolar solution oligo 15, as set out in
Table 1 below, in addition to oligos 10 and 11 as shown in Table
1.
[0282] Complexity determination: The protocol includes
quantification of doubly barcoded fragments prior to PCR. Doubly
barcoded fragment concentration was estimated in three ways:
quantitative PCR with a quenched fluorescent probe (oligo 19, as
set out in Table 1 below), dilution series endpoint PCR, and
quantification by next-generation sequencing. For the latter,
barcoded molecules were purified and serially diluted. Four
dilutions were amplified with oligo 6 and four versions of oligo
16, as set out in Table 1 below, containing different multiplexing
index sequences. The resulting products were mixed and sequenced
with 50-bp single-end reads on an Illumina MiSeq. Reads were
demultiplexed and unique barcodes at each dilution were counted.
When combined with the multiplexed library preparation strategy,
which enables further demultiplexing on the basis of an index in
the forward read, many samples can be quantified in a single MiSeq
run.
TABLE-US-00025 TABLE 1 Oligonucleotide sequences OLIGO NO.
Oligonucleotide Sequence 1 5'-/5Phos/NNN GTTCAGAGTTCTACAGTCCGACGATC
NNNNNNNNNNNNNNNN CC AGGAATAGTTATGTGCATTAATGAATGGCCGC-3' 2
5'-/5Phos/NNN CCTACACGACGCTCTTCCGATCT NNNNNNNNNNNNNNNN AC
AGGAATAGTTATGTGCATTAATGAATGGCCGC-3' 3 5'-/5Phos/NNN
CCTACACGACGCTCTTCCGATCT NNNNNNNNNNNNNNNN AC AATTCCTATCGTTCACGTCGTGT
CGCCATTTAGTGTCCAGTCTGA-3 4 5'-/5Phos/NNN CCTACACGACGCTCTTCCGATCT
NNNNNNNNNNNNNNNN CC AGGAATAGTTATGTGCATTAATGAATGG CGCC-3' 5
5'-CCATTCAT/ideoxyU/AATGCACA/ideoxyU/ AACTATTCC/3deoxyU/G*G-3' 6
5'-CCATTCAT/ideoxyU/AATGCACA/ideoxyU/ AACTATTCC/ideoxyU/G-3' 7
5'-ACACGACG/ideoxyU/GAACGA/ ideoxyU/AGGAAT/ideoxyU/G*T-3' 8
5'-CCGAGAATTCCA*T-3' 9 5'-/5Phos/TGGAATTCTCGG GTGCCAAGG-3' 10
5'-CAAGCAGAAGACGGCATACGAGAT (Index) GTGACTGGAGTT
CCTTGGCACCCGAGAATTCCA-3' 11 5'-
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATC*T-3' 12
5'-ACACTCTTTCCCTACACGAC GCTCTTCC-3' 13 5'-/5Phos/A*TC GGAAGAGC
ACACGTCT 14 5'-CAAGCAGAAGACGGCATACGAGAT (Index) GTGACTGGAGTTC
AGACGTGTGCTCTTCCGATC*T-3' 15 5'-
AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTT CTACAGTCCGA-3' 16
5'-CAAGCAGAAGACGGCATACGAGAT (Index) GTGACTGGAGTTC
AGACGTGTGCTCTTCCGATC CCATTCATTAATGCACATAACTATTCC-3' 17
5'-CCATTCATTAATGCACATAACTATTCCT GGNNNNNNNNNNNNNNNN
GATCGTCGGACTGTAGAACTCTGAAC T.sub.30VN-3' 18
5'-GCGGCCATTCATTAATGCACATAACTATTCCT GTNNNNNNNNNNNNNNNN
AGATCGGAAGAGCGTCGTGTAGG TrGrG+G-3' 19 5'-/56-FAM/CCT ACA CGA
/ZEN/CGC TCT TCC GAT CT/3IABkFQ/-3' 20 5'-NNN
CCTACACGACGCTCTTCCGATCT NNNNNNNNNNNNNNNN (Index) C
AGGAATAGTTATGTGCATTAATGAATGG CGCC-3' Key: /5Phos/ = 5` phosphate
group /ideoxyU/ = internal deoxyuracil base /3deoxyU/ = 3`
deoxyuracil base * = phosphorothioate linkage rG = riboG +G =
locked nucleic acid G N = mixture of A, T, G, and C V = mixture of
A, G, and C T.sub.30 = 30 consecutive Ts lcPCR = limited-cycle PCR
Index = 6-base Illumina TruSeq Small RNA multiplexing index
sequence /56-FAM/ = probe fluorophore /ZEN/ = probe quencher
/3IABkFQ/ = probe quencher
Example 2--Testing Barcode Fidelity
[0283] Example 2 illustrates experiments carried out to test
barcode fidelity. In general, a given barcode should be associated
with a single target molecule, i.e., barcode fidelity. With barcode
fidelity, every read tagged with that barcode should be derived
from that single target molecule and should contain nucleotide
sequence from that single target molecule alone.
[0284] Chimera formation during library preparation is problematic
to barcode fidelity when sequencing a mixed population of target
molecules. Once formed, chimeras are difficult to identify and
filter out, and can confound assembly or lead to reconstruction of
spurious sequences. Fortunately, the high coverage to which each
target molecule is sequenced renders the method tolerant to a
moderate level of chimera formation, in the same way that it
ameliorates the effect of NGS error rates. Assuming 20-fold
coverage at a chimera formation rate of 10%, half of the aligned
calls at a given locus are erroneous only 0.005% of the time.
[0285] To test barcode fidelity of the method with homologous
targets, a mixture of three linearized plasmids, each about 3 kb in
length with homologous but distinct inserts, were sequenced. Each
of the DNA plasmids, containing different mutants of the outer
membrane protein A (OmpA) gene of E. coli, were purified from E.
coli, linearized by restriction digestion, and mixed at known
ratios. The resulting sample contained molecules of three known
sequences, each at a different concentration. The target sequences
were highly homologous and, thus, susceptible to recombination
during PCR.
[0286] Following library preparation, sequencing, and
barcode-mediated read sorting, the reads associated with each
barcode were searched for short sequences unique to each target.
The experiments showed that in the majority of cases, the
contaminating reads were too few to confound analysis (see FIG. 2).
About 80% of barcodes were confidently assigned to one target.
Example 3--Sequencing Escherichia coli BL21
[0287] Genomic DNA was isolated from the model organism Escherichia
coli BL21 using a MasterPure.TM. DNA Purification Kit (Epicentre,
Madison, Wis.) and sheared into fragments of an average length of
about 3.5 kb using a HydroShear DNA Shearing System (Digilab,
Marlborough, Mass.). The fragment pool was converted to a
sequencing-ready library following the protocol described herein
and sequenced on a MiSeq sequencing instrument (Illumina, Inc., San
Diego, Calif.) with a 250 bp paired-end read reagent kit.
De-multiplexed reads were processed using a custom computational
pipeline, i.e., computer programs designed to process the
sequencing data and assemble the synthetic long reads. Groups of
reads sharing barcode sequences were assembled into long contiguous
sequences or "contigs," using the Velvet assembler, i.e., an
algorithm package designed to assemble contigs from sequence
information. See
(http://www.ebi.ac.uk/.about.zerbino/velvet/velvet_poster.pdf).
[0288] 743,538 paired-end read pairs were trimmed to remove
barcodes, spurious sequences, adapter sequences, and regions of low
quality. The read pairs were sorted into barcode-defined groups.
Barcode-defined groups were assembled with Velvet into 644 contigs,
wherein the contigs had lengths greater than 1,000 bp. The longest
contig was 4,423 bp, and the end of the distribution is in
concordance with the 3.5 kb average length of the sheared genomic
fragments, indicating that complete target molecule sequences were
reconstructed from some of the barcode groups using Velvet.
[0289] A histogram of barcode frequencies in the sequencing results
revealed the expected bimodal distribution. There is a bimodal
distribution because there are two types of barcodes: true barcodes
(seen many times each) and false barcodes caused by sequencing
errors (seen only a few times each). A peak at low numbers of times
seen corresponds to spurious barcodes resulting from sequencing
errors; these reads were discarded with no significant loss in
efficiency. A second peak, centered near 500 times seen per
barcode, corresponded to the true barcodes. This peak was much
broader than the ideal peak that would result from random selection
from an equal population of all barcodes, implying that PCR
amplification is biased, over-amplifying some targets at the
expense of others. This bias could be magnified by other parts of
the protocol.
[0290] Bias, in some aspects, can be reduced by modifications to
the protocol. For example, in some aspects, bias is reduced by
adding a linear amplification phase prior to exponential PCR, or by
optimizing PCR conditions (e.g., primer sequences, extension times,
annealing temperatures, etc). Still, given the low and rapidly
declining cost of sequencing, the current levels of bias do not
result in prohibitive inefficiency.
[0291] The relationship between the number of reads associated with
a barcode and the longest contig assembled from those reads
indicated that additional reads aid assembly (as expected) up to
about 1000 reads. However, not only do barcodes that are seen more
than 1000 times gain no extra advantage, the length of their
longest contigs drops off. In some aspects, this may be a result of
extra sequencing errors that confound assembly accumulating in
excess reads, or indicate that the most frequently seen barcodes
derive from spurious sequences.
[0292] The complexity bottleneck (a restriction on the number of
barcoded molecules) imposed upon the mixed DNA population by
dilution prior to PCR can be chosen for each experiment as a
function of the length of the target molecules and the number of
sequencing reads available. For example, in this experiment, the
true complexity bottleneck was estimated to have been on the order
of 1000 (about 700,000 reads divided by .about.500 reads per
barcode). Thus, the complexity (number of barcoded molecules) is
bottlenecked (restricted) prior to PCR to optimize sequence
assembly. If too many molecules are amplified in PCR, the
sequencing reads are spread out among them to the point that
full-length sequences cannot be assembled. If too few, then fewer
than an optimal number of sequences are assembled. The choice of
complexity depends on the number and length of reads to be
generated, the length of the target molecules, and whether barcode
pairing is used. In various aspects, determining the number of
barcoded molecules in a sample is done by qPCR, dilution-series
PCR, digital PCR, specific degradation of molecules lacking two
adapters followed by quantification, or sequencing.
[0293] A BLAST search of the assembled contigs against known
genomes confirmed that the majority of the contigs aligned to the
E. coli genome with high accuracy. Contigs of length greater than
250 bp were submitted to the query. The contigs that aligned with
the reference genome matched with 99.95% agreement, for an error
rate of 0.05%. It is notable that this 0.05% error rate represents
a ceiling on the error rate of the method, because the sequenced
strain may have accumulated mutations that differentiate it from
the reference, and because there is potential to optimize the
assembly algorithm for greater accuracy.
[0294] In every barcode pool alignment that was examined, about 80%
of the reads aligned within the same 3-4 kb region. The other 20%
aligned to other areas of the genome in a seemingly random manner,
likely as a result of intermolecular circularization during library
preparation. This fraction is reducible through optimization of the
circularization conditions, but this randomly scattered minority of
fragments does not typically confound assembly or other
applications of the method.
Example 4--Sequencing Geoglobus ahangari
[0295] Genomic DNA was isolated from the archaea Geoglobus ahangari
using the Masterpure.TM. DNA Purification Kit (Epicentre) and
sheared into fragments of an average length of 3.5 kb using a
HydroShear DNA Shearing System (Digilab). The fragment pool was
converted to a sequencing-ready library according to the protocol
provided above and sequenced on a MiSeq instrument (Illumina) with
a 250 bp paired-end read reagent kit. De-multiplexed reads were
processed using a custom computational pipeline, as described
herein. Groups of reads sharing barcode sequences were assembled
into contigs using the Velvet assembler. 2.3 million paired-end
read pairs were trimmed to remove barcodes, spurious sequences,
adapter sequences, and regions of low quality, and sorted into
barcode-defined groups. Using the Velvet assembler, the resultant
barcode groups were assembled into 1497 contigs of lengths greater
than 1,000 bp. The longest contig was 4,507 bp, and the end of the
distribution is in concordance with the 3.5 kb average length of
the sheared genomic fragments, indicating that Velvet was able to
reconstruct complete target molecule sequences from some of the
barcode groups.
[0296] Geoglobus ahangari contigs were used to improve an existing,
incomplete draft genome for this organism. The draft genome
contained 50 disconnected contigs. Long reads from the method
disclosed herein allowed the 50 disconnected contigs to be
collapsed into 30 contigs, containing no unresolved ("N") bases.
This experiment demonstrated that the long contigs derived from
methods of the disclosure dramatically improved the draft genome of
Geoglobus ahangari by resolving ambiguities in short-read
assemblies.
[0297] The bimodal distribution of barcode frequencies was less
pronounced in the Geoglobus data, indicating potentially more
severe PCR bias compared to the E. coli data. The true complexity
bottleneck is estimated to have been on the order of about 4000
(about 2.3 million reads divided by .about.500 reads per
barcode).
Example 5--Sequencing Tuberosum solanum
[0298] Genomic DNA was isolated from a doubled monoploid variety of
an important food crop, i.e., Tuberosum solanum (the potato), and
sheared into fragments of an average length of 3.5 kb using a
HydroShear DNA Shearing System (Digilab). The fragment pool was
converted to a sequencing-ready library according to the protocol
set out above and sequenced on a MiSeq instrument (Illumina) with a
250 bp paired-end read reagent kit. De-multiplexed reads were
processed using a custom computational pipeline, as described
herein. Groups of reads sharing barcode sequences were assembled
using the Velvet assembler.
[0299] 10.2 million paired-end read pairs were trimmed to remove
barcodes, spurious sequences, adapter sequences, and regions of low
quality, and sorted into barcode-defined groups. Using the Velvet
assembler, the resultant barcode groups were assembled into 1,508
contigs of length greater than 1,000 bp. The longest contig was
5,249 bp, and the end of the distribution was in concordance with
the 3.5 kb average length of the sheared genomic fragments,
indicating that Velvet was able to reconstruct complete target
molecule sequences from some of the barcode groups.
[0300] The sequencing results revealed the expected bimodal
distribution. The true complexity bottleneck was estimated to have
been on the order of about 4000 (about 10.2 million reads divided
by 3000 reads per barcode).
[0301] Assembled reads were analyzed further using bioinformatics.
A blind test was carried out because the experimenters did not have
access to the potato reference genome during contig assembly. The
potato contigs were aligned to an existing draft genome maintained
by the Potato Genome Consortium. Approximately 70-90% of the
contigs aligned to the reference genome, depending on the
stringency of the alignment parameters (minimum 98% agreement). The
high sequence agreement between the long contigs and the draft
genome highlighted the accuracy of contigs generated by methods of
the disclosure, in contrast to previously known long-read
technology. A Basic Local Alignment Search Tool (BLAST, NIH) search
returned hits to potato, as well as related organisms, including
tomato and nightshade. Potato is a tetraploid organism. Long reads,
such as those obtained by methods of the disclosure, are
instrumental to resolving the haplotype of each chromosome.
Example 6--Sequencing Escherichia coli Strain MG1655
[0302] Sequencing libraries were prepared from genomic DNA isolated
from E. coli strain MG1655. Genomic DNA was sheared and
size-selected to a range of about 5-10 kb. About 8 million 150 bp
paired-end read pairs were filtered and trimmed to remove barcodes,
adapter sequences, and regions of low quality and then sorted into
barcode-delineated groups, as described herein. Barcode pairing
resolved 1,186 distinct barcode pairs, whose read groups were
merged prior to assembly. Independent assembly of each group with
the SPAdes assembler (Bankevich et al., J. Computational Biology
19(5): 455-77, 2012) yielded 2,826 contigs of length greater than
1,000 bp.
[0303] To determine the fidelity of assembly, the largest contig
assembled from each barcode-defined group was aligned to the MG1655
reference genome (Hayashi et al., Mol. Syst. Biol. 2:0007, 2006).
Alignment of grouped reads to the reference genome showed a
non-uniform distribution of coverage across the fragment length,
with coverage dropping off along the length of the target sequence.
Barcode pairing reduced the impact of the coverage drop because
coverage from one barcode is high in the region where coverage from
its pair is low. Coverage is the number of short reads that align
to a given location on the long target sequence. Coverage drops
from one end of the target to the other, presumably because
circularization is less efficient for longer molecules. Coverage
from reads with the partner barcode is a mirror image: high on the
other end, and dropping toward the first end. The sum of the two
profiles is therefore relatively smoothed. This experiment showed
that assembly of longer molecules requires high average read
depths. Merging the paired read groups resulted in a smoother
distribution of coverage (see FIG. 1B.)
[0304] The length distribution of the assembled contigs had an N50
(half of the total assembled bases are in contigs greater than the
N50) of 6 kb and a maximum assembly length of 11.6 kb (see FIG.
1C). The error rate when contigs were aligned back to the reference
MG1655 genome was only about 0.1%. Thus, the experiment showed that
the method described herein was used to assemble contigs with an
N50 of 6 kb with about 99.9% accuracy.
Example 7--Sequencing Gelsemium sempervirens
[0305] Sequencing libraries were prepared from genomic DNA isolated
from Carolina jasmine (Gelsemium sempervirens), a plant with a
complex and previously unsequenced genome. 149,447 contigs longer
than 1 kb, with an N50 of 4 kb, were assembled. The assembled long
reads aligned with high stringency to a draft assembly of the
Gelsemium sempervirens genome, and increased the maximum scaffold
length from about 197,779 bp to about 365,589 bp. Thus, the
experiment showed that the method described herein was used to
assemble contigs with an N50 of 4 kb (see FIG. 1C), and was useful
in assembling a large portion of a previously unsequenced
genome.
Example 8--Library Preparation for Synthetic Long Read Assembly
from mRNA Samples
[0306] Full-length reverse transcripts were prepared with primers,
where the primers included oligo 17 and oligo 18, as set out in
Table 1 below, respectively. Barcoded full-length reverse
transcripts were then processed and sequenced, starting from
library quantification. The barcoded cDNA product was amplified,
broken, circularized, and prepared for sequencing. From mRNA
isolated from HCT116 and HepG2 cells, we assembled 28,689 and
16,929 synthetic reads, respectively, of lengths between 0.5 and
4.6 kb. Synthetic reads spanned multiple splice junctions, with a
median of 2.0 spanned junctions per synthetic read for both samples
and a maximum of 35 spanned junctions. Examination of the synthetic
reads revealed examples of differential splicing between the HCT116
and HepG2 cell lines, as well as a novel transcript in the HCT116
cell line.
Example 9--Multiplexed Sample Preparation
[0307] Two E. coli strains were isolated from each of twelve
recombination treatment populations (See e.g., Souza et al. Journal
of Evolutionary Biology 10:743-769, 1997). Genomic DNA was isolated
from each of the twenty-four strains, sheared, end-repaired, and
dT-tailed as described above in separate tubes. Twenty-four barcode
adapters (oligo 20, as set out in Table 1 below), identical except
for distinct 6-bp multiplexing index regions adjacent to the
barcode sequence, were prepared and ligated to the genomic
fragments as described above. Adapter-ligated DNA was PCR amplified
as above. Purified PCR products were quantified and equal amounts
were combined into a single mixture. This mixture was prepared for
sequencing following the other parts of the above protocol.
Sequencing reads were demultiplexed by project according to
standard 6-bp index read, then further demultiplexed by strain
according to the barcode-adjacent multiplexing index identified in
the forward read, sorted by barcode, and assembled in parallel. The
summed lengths of the synthetic reads longer than 1 kb exceeded
twofold genome coverage for sixteen out of the twenty-four strains,
with a median genome coverage of 2.3 fold and median N50 of 4.1
kb.
Example 10--Fragment Generation Based on Extension of Random
Primers
[0308] In some embodiments, fragments with randomly determined ends
are created by annealing primers of random or partially random
sequences. Each such primer anneals to a complimentary region of
the target molecule and is extended by a polymerase. In some cases,
the polymerase is capable of strand displacement. In some
instances, Bst polymerase is used. In some embodiments, phi29
polymerase is used. In some cases, Vent polymerase is used. In some
embodiments, this operation is preceded by linear or exponential
amplification of the targets. In some embodiments, the targets are
not amplified beforehand. In some cases, a mixture including
template molecules and random primers is melted at 95.degree. C.
and quenched to 0.degree. C. to allow primer annealing. Bst
polymerase can be added and the mixture can be slowly warmed to
65.degree. C. by ramping or stepping. In some cases, primers
complementary to the adapter ends of the target are present or are
added, and prime the single-stranded DNA synthesized following
random priming at its 3' end. Extension by a DNA polymerase
generates double-stranded DNA fragments with the known adapter end
sequence at one end and a random sequence from the interior of the
target molecule at the other end. In some embodiments, multiple
rounds of this linear amplification and fragment generation are
performed. In some embodiments, additional rounds are performed by
heating the mixture to e.g. 95.degree. C. to melt the
double-stranded DNA duplexes, cooling to promote random primer
annealing, and if necessary adding additional DNA polymerase. In
some embodiments, the target molecule adapters contain one or more
biotinylated nucleotides that allow them to specifically bind to
streptavidin-coated beads, so that the newly generated fragments
can be easily separated from the original targets between rounds of
amplification. In some embodiments, the random primers contain
defined sequences at their 5' end and random sequences at their 3'
end, so that the resulting ssDNA or dsDNA contains known sequences
at both ends. In some embodiments, the known sequences are the
same. In some embodiments, they are different. In some cases,
fragments are subsequently amplified by PCR using one or more
primers complementary to the known end sequences. In some
embodiments, DNA fragments created by linear or exponential
amplification contain known end sequences that are reverse
complements of each other and contain one or more deoxyuracil bases
in the 5' ends. A combination of uracil-DNA glycosylase (UDG) and
exonuclease VIII can then be used to remove the 5' ends, leaving
long single-stranded complimentary sequences that can anneal to
increase the efficiency of intramolecular circularization. In some
embodiments, treatment with UDG and exonuclease VIII is preceded by
treatment with Klenow fragment or a similar enzyme to remove
nontemplated deoxyadenosine bases added to the 3' ends during
extension. In some cases, the known end sequences contain sequences
that can be recognized by recombinase enzymes that circularize the
fragment by recombination. In some embodiments, circularization is
by blunt-end ligation.
[0309] In some cases, circularized fragments are fragmented by
mechanical or enzymatic (e.g. fragmentase, transposons) methods and
prepared for sequencing by ligating adapters and performing lcPCR
as described herein.
[0310] In some embodiments, circularized fragments are amplified by
rolling-circle amplification (RCA) or hyperbranching rolling-circle
amplification (HRCA). In some cases, RCA or HRCA is primed with
random primers or partially random primers. In some embodiments,
amplification is primed by one or more primers of defined sequence.
In some instances, amplification is performed in the presence of up
to 100% dUTP in place of dTTP, to allow the product be specifically
degraded later. In some embodiments, RCA or HCRA is followed by
mechanical or enzymatic fragmentation, adapter ligation, and PCR as
described herein. In some embodiments, RCA or HRCA is followed
directly by PCR or limited-cycle PCR. In some embodiments, PCR is
primed with one primer complementary to the defined sequence at the
5' end of the partially random primer used for RCA or HRCA, and a
second primer complementary to a sequence in the barcode adapter
proximal to the barcode sequence. In some embodiments, the PCR
primers are complementary to these sequences, but additionally
contain 5' extensions that add further sequences necessary for
sequencing. In some cases, RCA or HCRA products containing
deoxyuracil are subsequently degraded to enrich for PCR
products.
[0311] With reference to FIG. 8A, a mixture of target DNA
molecules, with barcode adapters attached to the ends according to
methods described herein, is prepared with the desired complexity
(number of distinct molecules). The barcode adapters contain an end
region of defined sequence (X), a degenerate barcode region (B)
that is different for every target molecule but defined for a given
individual molecule, and a defined region (I.sub.1) complementary
to some or all of one of the two eventual sequencing primers, such
as a standard sequencing primer (e.g., Illumina) or a custom
primer. Optionally, the molecules are amplified by linear or
exponential methods to create 10.sup.1-10.sup.5 copies of each
uniquely barcoded molecule. The target molecules are melted into
single-stranded DNA by heating or exposure to alkaline or other
denaturing conditions. One or more random or partially random
primers are then annealed along the length the target molecules by
rapid quenching to 0-4.degree. C. The primers depicted here are
partially random, with a random 3' region and a defined 5' region
(sequence Y).
[0312] Continuing with FIG. 8A and FIG. 8B, a strand-displacing DNA
polymerase, such as Bst DNA polymerase, is added to the
primer-annealed target DNA mixture. The temperature is ramped or
stepped up to 65.degree. C., and the polymerase extends each of the
random 3' primer ends annealed along the length of the target
molecule, displacing extended molecules in front of it as it goes
and releasing them into solution. One end of the newly synthesized
single-stranded DNA molecules is defined by the partially random
primer and contains the Y sequence followed by a sequence
complementary to the region of the target molecule to which a
specific primer from the degenerate mixture annealed. The other end
is defined by a sequence complementary to the end sequence of the
target molecule, which comprises A primer with a sequence
complementary to X is present in the mixture, and is designed with
an annealing temperature greater than 65.degree. C., allowing it to
anneal to the ends of the newly synthesized displaced molecules and
prime synthesis of the second strand, creating double-stranded DNA.
The result is a collection of target fragments, with no mechanical
or enzymatic shearing needed. If desired, multiple cycles of
melting, annealing, and strand-displacement amplification can be
performed to increase the yield of DNA. If desired, deoxyadenosine
overhangs added by the Bst polymerase in a template-independent
fashion can be removed by incubation with e.g. Klenow DNA
polymerase to create blunt-ended dsDNA.
[0313] Continuing with FIG. 8A and FIG. 8B, fragments synthesized
can be circularized by blunt-end ligation. Alternatively, to
improve circularization efficiency of long fragments, sticky-end
ligation can be performed, as shown here. If sequences X and Y in
the partially random primers and the second-strand primers are
synthesized so that they contain deoxyuracil bases, the USER enzyme
mix (UDG and endonuclease VIII) can excise the 5' ends of each
strand of the dsDNA to leave sticky ends of programmable length. If
X and Y are reverse-complements, the sticky ends will be
complementary, and will anneal to one another to promote
ligation.
[0314] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. It is not intended that the invention be limited by
the specific examples provided within the specification. While the
invention has been described with reference to the aforementioned
specification, the descriptions and illustrations of the
embodiments herein are not meant to be construed in a limiting
sense. Numerous variations, changes, and substitutions will now
occur to those skilled in the art without departing from the
invention. Furthermore, it shall be understood that all aspects of
the invention are not limited to the specific depictions,
configurations or relative proportions set forth herein which
depend upon a variety of conditions and variables. It should be
understood that various alternatives to the embodiments of the
invention described herein may be employed in practicing the
invention. It is therefore contemplated that the invention shall
also cover any such alternatives, modifications, variations or
equivalents. It is intended that the following claims define the
scope of the invention and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
Sequence CWU 1
1
35176DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(3)a, c, t, g, unknown
or othermodified_base(27)..(42)a, c, t, g, unknown or other
1nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnccaggaat agttatgtgc
60attaatgaat ggcgcc 76244DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(27)..(42)a, c, t, g, unknown or other
2nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nncc
44341DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(24)..(39)a, c, t, g, unknown
or other 3cctacacgac gctcttccga tctnnnnnnn nnnnnnnnnc c
41488DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(3)a, c, t, g, unknown
or othermodified_base(27)..(42)a, c, t, g, unknown or
othermodified_base(47)..(62)a, c, t, g, unknown or
othermodified_base(86)..(88)a, c, t, g, unknown or other
4nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnccggnnnn nnnnnnnnnn
60nnagatcgga agagcgtcgt gtaggnnn 88536DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(16)a, c, t, g, unknown or
othermodified_base(21)..(36)a, c, t, g, unknown or other
5nnnnnnnnnn nnnnnnccgg nnnnnnnnnn nnnnnn 36681DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(27)..(42)a, c, t, g, unknown or other
6nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnatcacgca ggaatagtta
60tgtgcattaa tgaatggcgc c 81749DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(27)..(42)a, c, t, g, unknown or other
7nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnatcacgc
49846DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(24)..(39)a, c, t, g, unknown
or other 8cctacacgac gctcttccga tctnnnnnnn nnnnnnnnna tcacgc
46923DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(16)a, c, t, g, unknown
or other 9nnnnnnnnnn nnnnnnatca cgc 231049DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(27)..(42)a, c, t, g, unknown or other
10nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnatcacgc
491198DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(3)a, c, t, g, unknown
or othermodified_base(27)..(42)a, c, t, g, unknown or
othermodified_base(57)..(72)a, c, t, g, unknown or
othermodified_base(96)..(98)a, c, t, g, unknown or other
11nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnatcacgcg cgtgatnnnn
60nnnnnnnnnn nnagatcgga agagcgtcgt gtaggnnn 981246DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(16)a, c, t, g, unknown or
othermodified_base(31)..(46)a, c, t, g, unknown or other
12nnnnnnnnnn nnnnnnatca cgcgcgtgat nnnnnnnnnn nnnnnn
461323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 13cctacacgac gctcttccga tct
231428DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 14aggaatagtt atgtgcatta atgaatgg
281579DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide5' phosphate groupmodified_base(1)..(3)a,
c, t, g, unknown or othermodified_base(30)..(45)a, c, t, g, unknown
or other 15nnngttcaga gttctacagt ccgacgatcn nnnnnnnnnn nnnnnccagg
aatagttatg 60tgcattaatg aatggccgc 791676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide5' phosphate groupmodified_base(1)..(3)a, c, t, g,
unknown or othermodified_base(27)..(42)a, c, t, g, unknown or other
16nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnacaggaat agttatgtgc
60attaatgaat ggccgc 761789DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide5' phosphate
groupmodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(27)..(42)a, c, t, g, unknown or other
17nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnacaattcc tatcgttcac
60gtcgtgtcgc catttagtgt ccagtctga 891876DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide5' phosphate groupmodified_base(1)..(3)a, c, t, g,
unknown or othermodified_base(27)..(42)a, c, t, g, unknown or other
18nnncctacac gacgctcttc cgatctnnnn nnnnnnnnnn nnccaggaat agttatgtgc
60attaatgaat ggcgcc 761930DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(9)..(9)Internal deoxyuracil
basemodified_base(18)..(18)Internal deoxyuracil
basemodified_base(28)..(28)3' deoxyuracil
basemisc_feature(29)..(30)Phosphorothioate linkage 19ccattcatua
atgcacauaa ctattccugg 302029DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(9)..(9)Internal deoxyuracil
basemodified_base(18)..(18)Internal deoxyuracil
basemodified_base(28)..(28)Internal deoxyuracil base 20ccattcatua
atgcacauaa ctattccug 292125DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(9)..(9)Internal deoxyuracil
basemodified_base(16)..(16)Internal deoxyuracil
basemodified_base(23)..(23)Internal deoxyuracil
basemisc_feature(24)..(25)Phosphorothioate linkage 21acacgacgug
aacgauagga atugt 252213DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemisc_feature(12)..(13)Phosphorothioate linkage
22ccgagaattc cat 132321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide5' phosphate group
23tggaattctc gggtgccaag g 212424DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 24caagcagaag
acggcatacg agat 242533DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 25gtgactggag
ttccttggca cccgagaatt cca 332658DNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemisc_feature(57)..(58)Phosphorothioate linkage
26aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct
582728DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 27acactctttc cctacacgac gctcttcc
282819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide5' phosphate
groupmisc_feature(1)..(2)Phosphorothioate linkage 28atcggaagag
cacacgtct 192934DNAArtificial SequenceDescription of Artificial
Sequence Synthetic
oligonucleotidemisc_feature(33)..(34)Phosphorothioate linkage
29gtgactggag ttcagacgtg tgctcttccg atct 343050DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30aatgatacgg cgaccaccga gatctacacg ttcagagttc
tacagtccga 503160DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 31gtgactggag ttcagacgtg
tgctcttccg atcccattca ttaatgcaca taactattcc 6032104DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
polynucleotidemodified_base(31)..(46)a, c, t, g, unknown or
othermodified_base(104)..(104)a, c, t, g, unknown or other
32ccattcatta atgcacataa ctattcctgg nnnnnnnnnn nnnnnngatc gtcggactgt
60agaactctga actttttttt tttttttttt tttttttttt ttvn
1043377DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotidemodified_base(35)..(50)a, c, t, g, unknown
or othermodified_base(75)..(76)Ribo-Gmodified_base(77)..(77)Locked
nucleic acid 33gcggccattc attaatgcac ataactattc ctgtnnnnnn
nnnnnnnnnn agatcggaag 60agcgtcgtgt aggtggg 773414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 34cgctcttccg atct 143533DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 35caggaatagt tatgtgcatt aatgaatggc gcc 33
* * * * *
References