U.S. patent application number 17/700088 was filed with the patent office on 2022-07-07 for methods and compositions for nucleic acid sequencing.
The applicant listed for this patent is ILLUMINA, INC.. Invention is credited to Sasan Amini, Igor Goryshin, Kevin L. Gunderson, Natasha Pignatelli, Frank J. Steemers.
Application Number | 20220213470 17/700088 |
Document ID | / |
Family ID | 1000006211117 |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220213470 |
Kind Code |
A1 |
Steemers; Frank J. ; et
al. |
July 7, 2022 |
METHODS AND COMPOSITIONS FOR NUCLEIC ACID SEQUENCING
Abstract
Embodiments of the present invention relate to sequencing
nucleic acids. In particular, embodiments of the methods and
compositions provided herein relate to preparing nucleic acid
templates and obtaining sequence data therefrom.
Inventors: |
Steemers; Frank J.;
(Encinitas, CA) ; Amini; Sasan; (Redwood City,
CA) ; Gunderson; Kevin L.; (Encinitas, CA) ;
Pignatelli; Natasha; (Berkeley, CA) ; Goryshin;
Igor; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ILLUMINA, INC. |
San Diego |
CA |
US |
|
|
Family ID: |
1000006211117 |
Appl. No.: |
17/700088 |
Filed: |
March 21, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16735348 |
Jan 6, 2020 |
11319534 |
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17700088 |
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14766089 |
Aug 5, 2015 |
10557133 |
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PCT/US2013/031023 |
Mar 13, 2013 |
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16735348 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12N 15/1065 20130101; C12Q 1/6806 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; C12Q 1/6869 20060101 C12Q001/6869; C12Q 1/6806 20060101
C12Q001/6806 |
Claims
1. A method for obtaining sequence information for a single cell,
comprising: generating indexed template nucleic acid fragments
derived from template nucleic acid of single cells, wherein the
indexed template nucleic acid fragments comprise a first index and
wherein the first index is associated with the template nucleic
acid derived from the single cell, combining the cells and
distributing the cells containing the indexed template nucleic acid
fragments into a plurality of vessels; providing a second index to
at least a portion of the indexed template nucleic acid fragments
in a vessel of the plurality of vessels to generate second indexed
template nucleic acid fragments comprising the first index and the
second index in the vessel; obtaining sequence data from the
indexed template nucleic acid fragments; and assembling a sequence
representation of the template nucleic acid from the sequence
data.
2. The method of claim 1, wherein the indexed template nucleic acid
is prepared by undergoing at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more rounds of compartmentalizing and indexing.
3. The method of claim 1, wherein generating the indexed template
nucleic acid fragments comprises using transposomes comprising the
first index to tagment the template nucleic acid.
4. The method of claim 1, wherein generating the indexed template
nucleic acid fragments comprises transposition, and where
subsequently the index is introduced using ligation
5. The method of claim 1, wherein the indexed nucleic acid
fragments of the cells are amplified with PCR.
6. The method of claim 1, wherein the vessel of the plurality of
vessels comprises at least 2 or more cells.
7. The method of claim 1, wherein the sequence representation
comprises haplotype information based at least in part on
assembling sequence reads from indexed template nucleic acid
fragments.
8. The method of claim 1, wherein another vessel of the plurality
of vessels comprises indexed template nucleic acid fragments
comprising the first index and a second index.
9. The method of claim 1, wherein the second index is associated
with the vessel.
10. A library of template nucleic acids, comprising: a tagmented
template nucleic acid comprising a plurality of transposons
inserted into a template nucleic acid using transposomes, wherein
at least some of the transposomes each comprise a first transposon
sequence and a second transposon sequence noncontiguous with said
first transposon sequence and a transposase, and wherein the
template nucleic acid is derived from a single cell.
11. The library of claim 10, wherein the tagmented template nucleic
acid from the cell is distributed into a vessel and assigned a
first index.
12. The library of claim 11, wherein the first index identifies the
sample.
13. The library of claim 12, wherein the first index sequence is
introduced using ligation.
14. The library of claim 12, wherein the index sequence is inserted
into the template nucleic acid using transposomes.
15. The library of claim 10, wherein the template nucleic acid is a
rolling circle amplification product.
16. The library of claim 10, wherein the template nucleic acid is
distributed in a well, on a microparticle or bead, or microfluidic
device.
17. The library of claim 10, wherein the tagmented template nucleic
acid is associated with or coupled to transposases of the
transposomes.
18. A method of obtaining sequence information from a single cell,
comprising obtaining a template nucleic acid of a cell comprising a
plurality of transposons inserted into said target nucleic acid
such that the contiguity of the template is retained,
compartmentalizing the nucleic acid of the cell comprising the
plurality of inserted transposons into a plurality of vessels,
generating compartment-specific indexed libraries from the
transposed nucleic acid targets and obtaining single cell sequence
information from the template nucleic acids from a plurality of
vessels.
19. The method of claim 18, wherein generating the
compartment-specific index is performed using transposition or
ligation.
20. The method of claim 18, wherein prior to generating the indexed
template nucleic acid fragments, the incorporation of a universal
primer site using transposition or ligation is used.
21. The method of claim 18, wherein generating the indexed template
nucleic acids fragments comprises (a) compartmentalizing the target
nucleic acid of cells into a plurality of first vessels; (b)
providing a first index to the target nucleic acid of each first
vessel, thereby obtaining a first indexed nucleic acid; (c)
combining the first indexed nucleic acids; (d) compartmentalizing
the first indexed template nucleic acids into a plurality of second
vessels; and (e) providing a second index to the first indexed
template nucleic of each second vessel, thereby obtaining a second
indexed nucleic acid.
22. The method of claim 21, wherein the indexed template nucleic
acids are generated by undergoing at least 2, 3, 4, 5, 6, 7, 8, 9,
10 or more rounds of compartmentalizing and indexing.
23. The method of claim 21, wherein providing the first index
comprises contacting the template nucleic acid with a plurality of
transposomes each comprising a transposase and a transposon
sequence comprising the first index under conditions such that at
least some of the transposon sequences insert into the template
nucleic acid.
24. The method of claim 21, wherein the first index is introduced
using ligation.
Description
FIELD OF THE INVENTION
[0001] Embodiments of the present invention relate to sequencing
nucleic acids. In particular, embodiments of the methods and
compositions provided herein relate to preparing nucleic acid
templates and obtaining sequence data therefrom.
BACKGROUND OF THE INVENTION
[0002] The detection of specific nucleic acid sequences present in
a biological sample has been used, for example, as a method for
identifying and classifying microorganisms, diagnosing infectious
diseases, detecting and characterizing genetic abnormalities,
identifying genetic changes associated with cancer, studying
genetic susceptibility to disease, and measuring response to
various types of treatment. A common technique for detecting
specific nucleic acid sequences in a biological sample is nucleic
acid sequencing.
[0003] Nucleic acid sequencing methodology has evolved
significantly from the chemical degradation methods used by Maxam
and Gilbert and the strand elongation methods used by Sanger. Today
several sequencing methodologies are in use which allow for the
parallel processing of nucleic acids all in a single sequencing
run. As such, the information generated from a single sequencing
run can be enormous.
SUMMARY OF THE INVENTION
[0004] Some embodiments of the methods and compositions provided
herein include a method of obtaining sequence information from a
target nucleic acid, said method comprising: (a) obtaining a
template nucleic acid comprising a plurality of transposomes
inserted into said target nucleic acid, wherein at least some of
the inserted transposomess each comprise a first transposon
sequence, a second transposon sequence noncontiguous with said
first transposon sequence, and a transposase associated with the
first transposon sequence and the second transposon sequence; (b)
compartmentalizing the template nucleic acid comprising said
plurality of inserted transposomes into each vessel of a plurality
of vessels; (c) removing the transposase from the template nucleic
acid; and (d) obtaining sequence information from the template
nucleic acid of each vessel.
[0005] In some embodiments, step (b) comprises providing each
vessel with an amount of template nucleic acid equal to about less
than one haploid equivalent, about equal than one haploid
equivalent, or more than one haploid equivalent of the target
nucleic acid,
[0006] In some embodiments, step (b) comprises providing each
vessel with an amount of template nucleic acid less than about one
haploid equivalent of the target nucleic acid.
[0007] In some embodiments, step (c) comprises a method selected
from the group consisting of adding a detergent, changing
temperature, changing pH, adding a protease, adding a chaperone,
and adding a polymerase.
[0008] In some embodiments, the first transposon sequence comprises
a first primer site and the second transposon sequences comprise a
second primer site.
[0009] In some embodiments, the first primer site further comprises
a first barcode and the second primer site further comprises a
second barcode.
[0010] In some embodiments, the first barcode and second barcode
are different.
[0011] In some embodiments, the target nucleic acid comprises an
amplified nucleic acid.
[0012] In some embodiments, the target nucleic acid is obtained by
enriching a plurality of nucleic acids for a sequence of interest
before or after transposition.
[0013] In some embodiments, step (a) further comprises enriching
the template nucleic acid for a sequence of interest.
[0014] In some embodiments, the target nucleic acid comprises
genomic DNA.
[0015] In some embodiments, step (d) further comprises assembling
from sequence data a representation of at least a portion of said
template nucleic acid from each vessel.
[0016] In some embodiments, the sequence information comprises
haplotype sequence information.
[0017] Some embodiments of the methods and compositions provided
herein include a method for preparing a library of template nucleic
acids to obtain sequence information from a target nucleic acid,
said method comprising: (a) preparing a template nucleic acid
comprising a plurality of transposomes inserted into said target
nucleic acid, wherein at least some of the inserted transposome
each comprise a first transposon sequence, a second transposon
sequence noncontiguous with said first transposon sequence, and a
transposase associated with the first transposon sequence and the
second transposon sequence; and (b) compartmentalizing the template
nucleic acid comprising said plurality of inserted transposomes
into each vessel of a plurality of vessels; and (c) removing the
transposase from the template nucleic acid.
[0018] In some embodiments, step (b) comprises providing each
vessel with an amount of template nucleic acid equal to less than
one haploid equivalent, about one equivalent, or more than one
equivalent of the target nucleic acid.
[0019] In some embodiments, step (b) comprises providing each
vessel with an amount of template nucleic acid less than about one
haploid equivalent of the target nucleic acid.
[0020] In some embodiments, step (c) comprises a method selected
from the group consisting of adding a detergent, changing
temperature, changing pH, adding a protease, adding a chaperone,
and adding a polymerase.
[0021] In some embodiments, the first transposon sequence comprises
a first primer site and the second transposon sequences comprise a
second primer site.
[0022] In some embodiments, the first primer site further comprises
a first barcode and the second primer site further comprises a
second barcode.
[0023] In some embodiments, the first barcode and second barcode
are different.
[0024] In some embodiments, the target nucleic acid comprises an
amplified nucleic acid.
[0025] In some embodiments, the target nucleic acid is obtained by
enriching a plurality of nucleic acids for a sequence of
interest.
[0026] In some embodiments, step (a) further comprises enriching
the template nucleic acid for a sequence of interest.
[0027] In some embodiments, the target nucleic acid comprises
genomic DNA.
[0028] In some embodiments, the sequence information comprises
haplotype sequence information.
[0029] Some embodiments of the methods and compositions provided
herein include a library of template nucleic acids prepared
according to any one of the foregoing methods.
[0030] Some embodiments of the methods and compositions provided
herein include a method of obtaining sequence information from a
target nucleic acid, said method comprising: (a) compartmentalizing
the target nucleic acid into a plurality of first vessels; (b)
providing a first index to the target nucleic acid of each first
vessel, thereby obtaining a first indexed nucleic acid; (c)
combining the first indexed nucleic acids; (d) compartmentalizing
the first indexed template nucleic acids into a plurality of second
vessels; (e) providing a second index to the first indexed template
nucleic of each second vessel, thereby obtaining a second indexed
nucleic acid; and (f) obtaining sequence information from the
second indexed nucleic acid of each second vessel.
[0031] In some embodiments, step (b) comprises contacting the
target nucleic acid with a plurality of transposomes each
comprising a transposase and a transposon sequence comprising the
first index under conditions such that at least some of the
transposon sequences insert into the target nucleic acid.
[0032] In some embodiments, step (b) comprises contacting the
target nucleic acid with a plurality of transposomes each
transposon comprising a first transposon sequence comprising a
first index, a second transposon sequence noncontiguous with said
first transposon sequence, and a transposase associated with the
first transposon sequence and the second transposon sequence.
[0033] In some embodiments, step (d) comprises removing the
transposase from the compartmentalized first indexed template
nucleic acids.
[0034] In some embodiments, the transposase is removed subsequent
to step (b).
[0035] In some embodiments, the transposase is removed prior to
step (f).
[0036] In some embodiments, removing the transposase comprises a
method selected from the group consisting of adding a detergent,
changing temperature, changing pH, adding a protease, adding a
chaperone, and adding a strand-displacing polymerase.
[0037] In some embodiments, the first transposon sequences
comprises a first primer site and the second transposon sequences
comprises a second primer site.
[0038] In some embodiments, the first primer site further comprises
a first barcode and the second primer site further comprises a
second barcode.
[0039] In some embodiments, the first barcode and second barcode
are different.
[0040] In some embodiments, step (b) comprises amplifying the
target nucleic acid with at least one primer comprising the first
index.
[0041] In some embodiments, step (b) comprises ligating the target
nucleic acid with at least one primer comprising the first
index.
[0042] In some embodiments, the first index provided to the target
nucleic acid of each first vessel is different.
[0043] In some embodiments, step (a) comprises providing each first
vessel with an amount of target nucleic acid greater than about one
or more haploid equivalents of the target nucleic acid.
[0044] In some embodiments, step (d) comprises providing each
vessel with an amount of the first indexed template nucleic acids
greater than about one or more haploid equivalents of the target
nucleic acid.
[0045] In some embodiments, step (e) comprises amplifying the first
indexed template nucleic with at least one primer comprising the
second index.
[0046] In some embodiments, step (e) comprises ligating the first
indexed template nucleic with at least one primer comprising the
second index.
[0047] In some embodiments, the second index provided to the first
indexed template nucleic of each second vessel is different.
[0048] In some embodiments, the target nucleic acid comprises an
amplified nucleic acid.
[0049] In some embodiments, the target nucleic acid is obtained by
enriching a plurality of nucleic acids for a sequence of
interest.
[0050] In some embodiments, the target nucleic acid comprises
genomic DNA.
[0051] In some embodiments, step (f) further comprises assembling
from sequence data a representation of at least a portion of said
template nucleic acid from each vessel.
[0052] Some embodiments of the methods and compositions provided
herein include a method preparing a library of template nucleic
acids to obtain sequence information from a target nucleic acid,
said method comprising: (a) compartmentalizing the target nucleic
acid into a plurality of first vessels; (b) providing a first index
to the target nucleic acid of each first vessel, thereby obtaining
a first indexed nucleic acid; (c) combining the first indexed
nucleic acids; (d) compartmentalizing the first indexed template
nucleic acids into a plurality of second vessels; and (e) providing
a second index to the first indexed template nucleic of each second
vessel, thereby obtaining a second indexed nucleic acid.
[0053] In some embodiments, step (b) comprises contacting the
target nucleic acid with a plurality of transposomes each
comprising a transposase and a transposon sequence comprising the
first index under conditions such that at least some of the
transposon sequences insert into the target nucleic acid.
[0054] In some embodiments, step (b) comprises contacting the
target nucleic acid with a plurality of transposomes each
transposon comprising a first transposon sequence comprising a
first index, a second transposon sequence noncontiguous with said
first transposon sequence, and a transposase associated with the
first transposon sequence and the second transposon sequence.
[0055] In some embodiments, step (d) comprises removing the
transposase from the compartmentalized first indexed template
nucleic acids.
[0056] In some embodiments, removing the transposase comprises a
method selected from the group consisting of adding a detergent,
changing temperature, changing pH, adding a protease, adding a
chaperone, and adding a polymerase.
[0057] In some embodiments, the first transposon sequences
comprises a first primer site and the second transposon sequences
comprises a second primer site.
[0058] In some embodiments, the first primer site further comprises
a first barcode and the second primer site further comprises a
second barcode.
[0059] In some embodiments, the first barcode and second barcode
are different.
[0060] In some embodiments, step (b) comprises amplifying the
target nucleic acid with at least one primer comprising the first
index.
[0061] In some embodiments, step (b) comprises ligating the target
nucleic acid with at least one primer comprising the first
index.
[0062] In some embodiments, the first index provided to the target
nucleic acid of each first vessel is different.
[0063] In some embodiments, step (a) comprises providing each first
vessel with an amount of target nucleic acid greater than about one
or more haploid equivalents of the target nucleic acid.
[0064] In some embodiments, step (d) comprises providing each
vessel with an amount of the first indexed template nucleic acids
greater than about one or more haploid equivalents of the target
nucleic acid.
[0065] In some embodiments, step (e) comprises amplifying the first
indexed template nucleic with at least one primer comprising the
second index.
[0066] In some embodiments, step (e) comprises ligating the first
indexed template nucleic with at least one primer comprising the
second index.
[0067] In some embodiments, the second index provided to the first
indexed template nucleic of each second vessel is different.
[0068] In some embodiments, the target nucleic acid comprises an
amplified nucleic acid.
[0069] In some embodiments, the target nucleic acid is obtained by
enriching a plurality of nucleic acids for a sequence of interest
either before or after transposition.
[0070] In some embodiments, the target nucleic acid comprises
genomic DNA.
[0071] Some embodiments of the methods and compositions provided
herein include a library of template nucleic acids prepared
according to any one of the foregoing methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIG. 1 depicts a schematic of a transposome comprising a
dimeric transposase and two non-contiguous transposon sequences,
and a transposome comprising a dimeric transposase and a contiguous
transposon sequence.
[0073] FIG. 2 depicts a method of preparing a transposome with a
linker comprising a complementary double-stranded sequence.
[0074] FIG. 3 depicts an embodiment of making a template library
using transposomes comprising transposon sequences comprising a
single stranded linker coupling the two transposon sequences in
each transposome in a 5'-5' orientation. Sequences are extended
using primers
[0075] FIG. 4 depicts a scheme for preparing template nucleic acids
to obtain sequence information in which a target nucleic acid is
compartmentalized into 96 tubes, indexed by insertion of Tn5
derived transposons, indexed nucleic acids are combined, and
further compartmentalized into 96 tubes, further indexed by
amplification, the twice indexed nucleic acids may then be
combined.
[0076] FIG. 5 depicts a schematic embodiment for obtaining
haplotype sequence information in which a template nucleic acid is
indexed with the barcode of a transposon, and with a primer. A
template nucleic acid is prepared by insertion of a looped
transposon into a target nucleic acid. The template nucleic acid is
diluted into compartments. The template nucleic acid of each
compartment is indexed by amplification with a primer. Indexed
template nucleic acids are sequenced, aligned, and sequence
representation is obtained.
[0077] FIG. 6 depicts a scheme that includes preparing a target
nucleic acid using matepair and rolling circle amplification,
followed by insertion of transposomes into the target nucleic acid,
dilution of the target nucleic acid to obtain haplotype
information, removal of the transposase by addition of SDS,
generation of shotgun libraries, indexing and sequencing.
[0078] FIG. 7 depicts a scheme that includes preparing a target
nucleic acid using hairpin transposition and rolling circle
amplification, followed by insertion of transposomes into the
target nucleic acid, dilution of the target nucleic acid, removal
of the transposase by addition of SDS, generation of shotgun
libraries, indexing and sequencing to obtain haplotype
information.
[0079] FIG. 8 depicts an example scheme for generation of mate pair
libraries.
[0080] FIG. 9 depicts an example scheme for generation of mate pair
libraries.
[0081] FIG. 10 is a graph depicting a model of error rate in
sequence information for the number of times a particular sequence
associated with a barcode is sequenced.
[0082] FIG. 11 depicts images of agarose gels showing
oligonucleotides linked with 5'-5' bisoxyamine coupling, in which
looped precursor transposons are indicated by the dimer band.
[0083] FIG. 12 is an image of an agarose gel showing the apparent
molecular weight of a transposed target nucleic acid associated
with transposase (left lane), and without transposase (+0.1% SDS,
middle lane).
[0084] FIG. 13 summarizes that haplotype blocks up to 100 kb were
observed for samples in which transposase was removed by SDS
post-dilution.
[0085] FIG. 14 depicts a graph showing the frequencies of
sequencing reads for particular distances between neighboring
aligned reads for template nucleic acids prepared by adding SDS to
remove transposase before dilution to obtain haplotype information,
or after dilution to obtain haplotype information.
[0086] FIG. 15 shows a graph of barcode indices and proportion of
reads and demonstrates that all 9216 different compartments in a
96.times.96 indexing scheme were observed.
[0087] FIG. 16 depicts a Pile up analysis of haplotype information
obtained using transposomes comprising Mu.
DETAILED DESCRIPTION
[0088] Embodiments of the present invention relate to sequencing
nucleic acids. In particular, embodiments of the methods and
compositions provided herein relate to preparing nucleic acid
templates and obtaining sequence data therefrom. Methods and
compositions provided herein are related to the methods and
compositions provided in U.S. Patent Application Pub. No.
2012/0208705, U.S. Patent Application Pub. No. 2012/0208724 and
Int. Patent Application Pub. No. WO 2012/061832, each of which is
incorporated by reference in its entirety. Some embodiments of the
present invention relate to preparing templates to obtain haplotype
sequence information from a target nucleic acid, and obtaining
haplotype sequence information from such templates. More
embodiments relate to preparing templates to obtain sequence
information from a strand of a double-stranded target nucleic acid,
and obtaining sequence information from such templates. Particular
embodiments provided herein relate to the use of integrases, for
example transposases, to maintain physical proximity of associated
ends of fragmented nucleic acids; and to the use of virtual
compartments to enable the use of high concentrations of nucleic
acids.
[0089] Obtaining haplotype information from a target nucleic acid
includes distinguishing between different alleles (e.g., SNPs,
genetic anomalies, etc.) in a target nucleic acid. Such methods are
useful to characterize different alleles in a target nucleic acid,
and to reduce the error rate in sequence information. Generally,
methods to obtain haplotype sequence information include obtaining
sequence information for a portion of a template nucleic acid. In
one embodiment, a template nucleic acid can be diluted and sequence
information obtained from an amount of template nucleic acid
equivalent to about a haplotype of the target nucleic acid.
[0090] In further embodiments, a template nucleic acid can be
compartmentalized such that multiple copies of a chromosome can be
present in the same compartment, as a result of dual or multiple
indexing provided herein, a haplotype can still also be determined.
In other words, a template nucleic acid can be prepared using
virtual compartments. In such embodiments, a nucleic acid can be
distributed between several first compartments, providing a first
index to the nucleic acid of each compartment, combining the
nucleic acids, distributing the nucleic acid between several second
compartments, and providing a second index to the nucleic acid of
each compartment. Advantageously, such indexing enables haplotype
information to be obtained at higher concentrations of nucleic acid
compared to the mere dilution of a nucleic acid in a single
compartment to an amount equivalent to a haplotype of the nucleic
acid.
[0091] In some embodiments provided herein, template libraries are
prepared using transposomes. In some such libraries, the target
nucleic acid may be fragmented. Accordingly, some embodiments
provided herein relate to methods for maintaining sequence
information for the physical contiguity of adjacent fragments. Such
methods include the use of integrases to maintain the association
of template nucleic acid fragments adjacent in the target nucleic
acid. Advantageously, such use of integrases to maintain physical
proximity of fragmented nucleic acids increases the likelihood that
fragmented nucleic acids from the same original molecule, e,g,
chromosome, will occur in the same compartment.
[0092] Other embodiments provided herein relate to obtaining
sequence information from each strand of a nucleic acid which can
be useful to reduce the error rate in sequencing information.
Methods to prepare libraries of template nucleic acids for
obtaining sequence information from each strand of a nucleic acid
can be prepared such that each strand can be distinguished, and the
products of each strand can also be distinguished.
[0093] Some of the methods provided herein include methods of
analyzing nucleic acids. Such methods include preparing a library
of template nucleic acids of a target nucleic acid, obtaining
sequence data from the library of template nucleic acids, and
assembling a sequence representation of the target nucleic acid
from such sequence data.
[0094] Generally, the methods and compositions provided herein are
related to the methods and compositions provided in U.S. Patent
Application Pub. No. 2012/0208705, U.S. Patent Application Pub. No.
2012/0208724 and Int. Patent Application Pub. No. WO 2012/061832,
each of which is incorporated by reference in its entirety. The
methods provided herein relate to the use of transposomes useful to
insert features into a target nucleic acid. Such features including
fragmentation sites, primer sites, barcodes, affinity tags,
reporter moieties, etc.
[0095] In a method useful with the embodiments provided herein, a
library of template nucleic acids is prepared from a target nucleic
acid. The library is prepared by inserting a plurality of unique
barcodes throughout the target nucleic acid. In some embodiments,
each barcode includes a first barcode sequence and a second barcode
sequence, having a fragmentation site disposed therebetween. The
first barcode sequence and second barcode sequence can be
identified or designated to be paired with one another. The pairing
can be informative so that a first barcode is associated with a
second barcode. Advantageously, the paired barcode sequences can be
used to assemble sequencing data from the library of template
nucleic acids. For example, identifying a first template nucleic
acid comprising a first barcode sequence and a second template
nucleic acid comprising a second barcode sequence that is paired
with the first indicates that the first and second template nucleic
acids represent sequences adjacent to one another in a sequence
representation of the target nucleic acid. Such methods can be used
to assemble a sequence representation of a target nucleic acid de
novo, without the requirement of a reference genome.
Definitions
[0096] As used herein the term "nucleic acid" and/or
"oligonucleotide" and/or grammatical equivalents thereof can refer
to at least two nucleotide monomers linked together. A nucleic acid
can generally contain phosphodiester bonds; however, in some
embodiments, nucleic acid analogs may have other types of
backbones, comprising, for example, phosphoramide (Beaucage, et
al., Tetrahedron, 49:1925 (1993); Letsinger, J. Org. Chem., 35:3800
(1970); Sprinzl, et al., Eur. J. Biochem., 81:579 (1977);
Letsinger, et al., Nucl. Acids Res., 14:3487 (1986); Sawai, et al.,
Chem. Lett., 805 (1984), Letsinger, et al., J. Am. Chem. Soc.,
110:4470 (1988); and Pauwels, et al., Chemica Scripta, 26:141
(1986)), phosphorothioate (Mag, et al., Nucleic Acids Res., 19:1437
(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu, et
al., J. Am. Chem. Soc., 111:2321 (1989), O-methylphosphoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid
backbones and linkages (see Egholm, J. Am. Chem. Soc., 114:1895
(1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992);
Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207
(1996)).
[0097] Other analog nucleic acids include those with positive
backbones (Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097
(1995)); non-ionic backbones (U.S. Pat. Nos. 5,386,023; 5,637,684;
5,602,240; 5,216,141; and 4,469,863; Kiedrowshi, et al., Angew.
Chem. Intl. Ed. English, 30:423 (1991); Letsinger, et al., J. Am.
Chem. Soc., 110:4470 (1988); Letsinger, et al., Nucleosides &
Nucleotides, 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series
580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook; Mesmaeker, et al., Bioorganic &
Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J. Biomolecular
NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) and non-ribose
(U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC
Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed, Y, S. Sanghui and P. Dan Coo). Nucleic acids may
also contain one or more carbocyclic sugars (see Jenkins, et al.,
Chem. Soc. Rev., (1995) pp. 169 176).
[0098] Modifications of the ribose-phosphate backbone may be done
to facilitate the addition of additional moieties such as labels,
or to increase the stability of such molecules under certain
conditions. In addition, mixtures of naturally occurring nucleic
acids and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. The nucleic acids may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The nucleic acid
may be DNA, for example, genomic or cDNA, RNA or a hybrid, from
single cells, multiple cells, or from multiple species, as with
metagenomic samples, such as from environmental samples, further
from mixed samples for example mixed tissue samples or mixed
samples for different individuals of the same species, disease
samples such as cancer related nucleic acids, and the like. A
nucleic acid can contain any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xanthanine,
hypoxanthanine, isocytosine, isoguanine, and base analogs such as
nitropyrrole (including 3-nitropyrrole) and nitroindole (including
5-nitroindole), etc.
[0099] In some embodiments, a nucleic acid can include at least one
promiscuous base. Promiscuous bases can base-pair with more than
one different type of base. In some embodiments, a promiscuous base
can base-pair with at least two different types of bases and no
more than three different types of bases. An example of a
promiscuous base includes inosine that may pair with adenine,
thymine, or cytosine. Other examples include hypoxanthine,
5-nitroindole, acylic 5-nitroindole, 4-nitropyrazole,
4-nitroimidazole and 3-nitropyrrole (Loakes et al., Nucleic Acid
Res. 22:4039 (1994); Van Aerschot et al., Nucleic Acid Res. 23:4363
(1995); Nichols et al., Nature 369:492 (1994); Bergstrom et al.,
Nucleic Acid Res. 25:1935 (1997); Loakes et al., Nucleic Acid Res.
23:2361 (1995); Loakes et al., J. Mol. Biol. 270:426 (1997); and
Fotin et al., Nucleic Acid Res. 26:1515 (1998)). Promiscuous bases
that can base-pair with at least three, four or more types of bases
can also be used.
[0100] As used herein, the term "nucleotide analog" and/or
grammatical equivalents thereof can refer to synthetic analogs
having modified nucleotide base portions, modified pentose
portions, and/or modified phosphate portions, and, in the case of
polynucleotides, modified internucleotide linkages, as generally
described elsewhere (e.g., Scheit, Nucleotide Analogs, John Wiley,
New York, 1980; Englisch, Angew. Chem. Int. Ed. Engl. 30:613-29,
1991; Agarwal, Protocols for Polynucleotides and Analogs, Humana
Press, 1994; and S. Verma and F. Eckstein, Ann. Rev. Biochem.
67:99-134, 1998). Generally, modified phosphate portions comprise
analogs of phosphate wherein the phosphorous atom is in the +5
oxidation state and one or more of the oxygen atoms is replaced
with a non-oxygen moiety, e.g., sulfur. Exemplary phosphate analogs
include but are not limited to phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselcnoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate,
boronophosphates, including associated counterions, e.g., H.sup.+,
NH.sub.4.sup.+, Na.sup.+, if such counterions are present. Example
modified nucleotide base portions include but are not limited to
5-methylcytosine (5mC); C-5-propynyl analogs, including but not
limited to, C-5 propynyl-C and C-5 propynyl-U; 2,6-diaminopurine,
also known as 2-amino adenine or 2-amino-dA); hypoxanthine,
pseudouridine, 2-thiopyrimidine, isocytosine (isoC), 5-methyl isoC,
and isoguanine (isoG; see, e.g., U.S. Pat. No. 5,432,272).
Exemplary modified pentose portions include hut are not limited to,
locked nucleic acid (LNA) analogs including without limitation
Bz-A-LNA, 5-Me-Bz-C-LNA, dmf-G-LNA, and T-LNA (see, e.g., The Glen
Report, 16(2):5, 2003; Koshkin et al., Tetrahedron 54:3607-30,
1998), and 2'- or 3'-modifications where the 2'- or 3'-position is
hydrogen, hydroxy, alkoxy (e.g., methoxy, ethoxy, allyloxy,
isopropoxy, butoxy, isobutoxy and phenoxy), azido, amino,
alkylamino, fluoro, chloro, or bromo. Modified internucleotide
linkages include phosphate analogs, analogs having achiral and
uncharged intersubunit linkages (e.g., Sterchak, E. P. et al.,
Organic Chem., 52:4202, 1987), and uncharged morpholino-based
polymers having achiral intersubunit linkages (see, e.g., U.S. Pat.
No. 5,034,506). Some internucleotide linkage analogs include
morpholidate, acetal, and polyamide-linked heterocycles. In one
class of nucleotide analogs, known as peptide nucleic acids,
including pseudocomplementary peptide nucleic acids ("PNA"), a
conventional sugar and internucleotide linkage has been replaced
with a 2-aminoethylglycine amide backbone polymer (see, e.g.,
Nielsen et al., Science, 254:1497-1500, 1991; Egholm et al., J. Am.
Chem. Soc., 114: 1895-1897 1992; Demidov et al., Proc. Natl. Acad.
Sci. 99:5953-58, 2002; Peptide Nucleic Acids: Protocols and
Applications, Nielsen, ed., Horizon Bioscience, 2004),
[0101] As used herein, the term "sequencing read" and/or
grammatical equivalents thereof can refer to a repetitive process
of physical or chemical steps that is carried out to obtain signals
indicative of the order of monomers in a polymer. The signals can
be indicative of an order of monomers at single monomer resolution
or lower resolution. In particular embodiments, the steps can be
initiated on a nucleic acid target and carried out to obtain
signals indicative of the order of bases in the nucleic acid
target. The process can be carried out to its typical completion,
which is usually defined by the point at which signals from the
process can no longer distinguish bases of the target with a
reasonable level of certainty. If desired, completion can occur
earlier, for example, once a desired amount of sequence information
has been obtained. A sequencing read can be carried out on a single
target nucleic acid molecule or simultaneously on a population of
target nucleic acid molecules having the same sequence, or
simultaneously on a population of target nucleic acids having
different sequences. In some embodiments, a sequencing read is
terminated when signals are no longer obtained from one or more
target nucleic acid molecules from which signal acquisition was
initiated. For example, a sequencing read can be initiated for one
or more target nucleic acid molecules that are present on a solid
phase substrate and terminated upon removal of the one or more
target nucleic acid molecules from the substrate. Sequencing can be
terminated by otherwise ceasing detection of the target nucleic
acids that were present on the substrate when the sequencing run
was initiated.
[0102] As used herein, the term "sequencing representation" and/or
grammatical equivalents thereof can refer to information that
signifies the order and type of monomeric units in the polymer. For
example, the information can indicate the order and type of
nucleotides in a nucleic acid. The information can be in any of a
variety of formats including, for example, a depiction, image,
electronic medium, series of symbols, series of numbers, series of
letters, series of colors, etc. The information can be at single
monomer resolution or at lower resolution. An exemplary polymer is
a nucleic acid, such as DNA or RNA, having nucleotide units. A
series of "A," "T," "G," and "C" letters is a well-known sequence
representation for DNA that can be correlated, at single nucleotide
resolution, with the actual sequence of a DNA molecule. Other
exemplary polymers are proteins having amino acid units and
polysaccharides having saccharide units.
[0103] As used herein the term "at least a portion" and/or
grammatical equivalents thereof can refer to any fraction of a
whole amount. For example, "at least a portion" can refer to at
least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99%, 99.9% or 100% of a whole amount.
Transposomes
[0104] A "transposome" comprises an integration enzyme such as an
integrase or transposase, and a nucleic acid comprising an
integration recognition site, such as a transposase recognition
site. In embodiments provided herein, the transposase can form a
functional complex with a transposase recognition site that is
capable of catalyzing a transposition reaction. The transposase may
bind to the transposase recognition site and insert the transposase
recognition site into a target nucleic acid in a process sometimes
termed "tagmentation". In some such insertion events, one strand of
the transposase recognition site may be transferred into the target
nucleic acid. FIG. 1 depicts two examples of transposomes. In one
example, a transposome (10) comprises a dimeric transposase
comprising two subunits (20), and two non-contiguous transposon
sequences (30). In another example, a transposome (50) comprises a
transposase comprises a dimeric transposase comprising two subunits
(60), and a contiguous transposon sequence (70).
[0105] Some embodiments can include the use of a hyperactive Tn5
transposase and a Tn5-type transposase recognition site (Goryshin
and Reznikoff, J. Biol. Chem., 273:7367 (1998)), or MuA transposase
and a Mu transposase recognition site comprising R1 and R2 end
sequences (Mizuuchi, K., Cell, 35: 785, 1983; Savilahti, H, et al.,
EMBO J., 14: 4893, 1995). ME sequences can also be used as
optimized by a skilled artisan.
[0106] More examples of transposition systems that can be used with
certain embodiments of the compositions and methods provided herein
include Staphylococcus aureus Tn552 (Colegio et al., J. Bacteriol.,
183: 2384-8, 2001; Kirby C et al., Mol. Microbiol., 43: 173-86,
2002), Ty1 (Devine & Boeke, Nucleic Acids Res., 22: 3765-72,
1994 and International Publication WO 95/23875), Transposon Tn7
(Craig, N L, Science. 271: 1512, 1996; Craig, N L, Review in: Curr
Top Microbiol Immunol., 204:27-48, 1996), Tn/O and IS10 (Kleckner
N, et al., Curr Top Microbiol Immunol., 204:49-82, 1996), Mariner
transposase (Lampe D J, et al., EMBO J., 15: 5470-9, 1996), Tc1
(Plasterk R H, Curr. Topics Microbiol. Immunol., 204: 125-43,
1996), P Element (Gloor, G B, Methods Mol. Biol., 260: 97-114,
2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265:18829-32,
1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr.
Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses (Brown, et
al., Proc Natl Acad Sci USA, 86:2525-9, 1989), and retrotransposon
of yeast (Boeke & Corces, Annu Rev Microbiol. 43:403-34, 1989).
More examples include IS5, Tn10, Tn903, IS911, and engineered
versions of transposase family enzymes (Zhang et al., (2009) PLoS
Genet. 5:e1000689. Epub 2009 Oct. 16; Wilson C. et al (2007) J.
Microbiol. Methods 71:332-5).
[0107] More examples of integrases that may be used with the
methods and compositions provided herein include retroviral
integrases and integrase recognition sequences for such retroviral
integrases, such as integrases from HIV-1, HIV-2, SIV, PFV-1,
RSV.
Transposon Sequences
[0108] Some embodiments of the compositions and methods provided
herein include transposon sequences. In some embodiments, a
transposon sequence includes at least one transposase recognition
site. In some embodiments, a transposon sequence includes at least
one transposase recognition site and at least one barcode.
Transposon sequences useful with the methods and compositions
provided herein are provided in U.S. Patent Application Pub. No.
2012/0208705, U.S. Patent Application Pub. No. 2012/0208724 and
Int. Patent Application Pub. No. WO 2012/061832, each of which is
incorporated by reference in its entirety. In some embodiments, a
transposon sequence includes a first transposase recognition site,
a second transposase recognition site, and a barcode or barcodes
disposed therebetween.
Transposomes with Non-Contiguous Transposon Sequences
[0109] Some transposomes provided herein include a transposase
comprising two transposon sequences. In some such embodiments, the
two transposon sequences are not linked to one another, in other
words, the transposon sequences are non-contiguous with one
another. Examples of such transposomes are known in the art, see
e.g., U.S. Patent Application Pub. No. 2010/0120098, the disclosure
of which is incorporated herein by reference in its entirety. FIG.
1 depicts an example transposome (10) comprising a dimeric
transposase (20) and two transposon sequences (30).
Looped Structures
[0110] In some embodiments, a transposome comprises a transposon
sequence nucleic acid that binds two transposase subunits to form a
"looped complex" or a "looped transposome." In essence, a
transposase complex with contiguous transposons. FIG. 1 depicts an
example transposome (50) comprising a dimeric transposase (60) and
a transposon sequence (70). Looped complexes can ensure that
transposons are inserted into target DNA while maintaining ordering
information of the original target DNA and without fragmenting the
target DNA. As will be appreciated, looped structures may insert
primers, barcodes, indexes and the like into a target nucleic acid,
while maintaining physical connectivity of the target nucleic acid.
In some embodiments, the transposon sequence of a looped
transposome can include a fragmentation site such that the
transposon sequence can be fragmented to create a transposome
comprising two transposon sequences. Such transposomes are useful
to ensuring that neighboring target DNA fragments, in which the
transposons insert, receive code combinations that can be
unambiguously assembled at a later stage of the assay.
Barcodes
[0111] Generally, a barcode can include one or more nucleotide
sequences that can be used to identify one or more particular
nucleic acids. The barcode can be an artificial sequence, or can be
a naturally occurring sequence generated during transposition, such
as identical flanking genomic DNA sequences (g-codes) at the end of
formerly juxtaposed DNA fragments. A barcode can comprise at least
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or more consecutive nucleotides. In sonic embodiments, a
barcode comprises at least about 10, 20, 30, 40, 50, 60, 70 80, 90,
100 or more consecutive nucleotides. In some embodiments, at least
a portion of the barcodes in a population of nucleic acids
comprising barcodes is different. In some embodiments, at least
about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of the
barcodes are different. In more such embodiments, all of the
barcodes are different. The diversity of different barcodes in a
population of nucleic acids comprising barcodes can be randomly
generated or non-randomly generated.
[0112] In some embodiments, a transposon sequence comprises at
least one barcode. In some embodiments, such as transposomes
comprising two non-contiguous transposon sequences, the first
transposon sequence comprises a first barcode, and the second
transposon sequence comprises a second barcode. In some
embodiments, such as in looped transposomes, a transposon sequence
comprises a barcode comprising a first barcode sequence and a
second barcode sequence. In some of the foregoing embodiments, the
first barcode sequence can be identified or designated to be paired
with the second barcode sequence. For example, a known first
barcode sequence can be known to be paired with a known second
barcode sequence using a reference table comprising a plurality of
first and second bar code sequences known to be paired to one
another.
[0113] In another example, the first barcode sequence can comprise
the same sequence as the second barcode sequence. In another
example, the first barcode sequence can comprise the reverse
complement of the second barcode sequence. In some embodiments, the
first barcode sequence and the second barcode sequence are
different. The first and second barcode sequences may comprise a
bi-code.
[0114] In some embodiments of compositions and methods described
herein, barcodes are used in the preparation of template nucleic
acids. As will be understood, the vast number of available barcodes
permits each template nucleic acid molecule to comprise a unique
identification. Unique identification of each molecule in a mixture
of template nucleic acids can be used in several applications. For
example, uniquely identified molecules can be applied to identify
individual nucleic acid molecules, in samples having multiple
chromosomes, in genomes, in cells, in cell types, in cell disease
states, and in species, for example, in haplotype sequencing, in
parental allele discrimination, in metagenomic sequencing, and in
sample sequencing of a genome.
Linkers
[0115] Some embodiments comprising looped transposomes where a
transposase is complexed with contiguous transposons include
transposon sequences comprising a first barcode sequence and a
second barcode sequence having a linker disposed therebetween. In
other embodiments, the linker can be absent, or can be the
sugar-phosphate backbone that connects one nucleotide to another.
The linker can comprise, for example, one or more of a nucleotide,
a nucleic acid, a non-nucleotide chemical moiety, a nucleotide
analogue, amino acid, peptide, polypeptide, or protein. In
preferred embodiments, a linker comprises a nucleic acid. The
linker can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In
some embodiments, a linker can comprise at least about 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more
nucleotides.
[0116] In some embodiments, a linker can be amplifiable for example
by PCR, rolling circle amplification, strand displacement
amplification, and the like. In other embodiments, a linker can
comprise non-amplifiable moieties. Examples of non-amplifiable
linkers include organic chemical linkers such as alkyl, propyl,
PEG; non-natural bases such as IsoC, isoG; or any group that does
not amplify in DNA-based amplification schemes. For example,
transposons containing isoC, isoG pairs can be amplified with dNTPs
mixtures lacking a complementary isoG and isoC, ensuring that no
amplification occurs across the inserted transposons.
[0117] In some embodiments, the linker comprises a single-stranded
nucleic acid. In some embodiments, the linker couples transposon
sequences in a 5'-3' orientation, a 5'-5' orientation, or a 3'-3'
orientation.
Fragmentation Sites
[0118] In some embodiments comprising looped transposomes the
linker can comprise a fragmentation site. A fragmentation site can
be used to cleave the physical, but not the informational
association between a first barcode sequence and a second barcode
sequence. Cleavage may be by biochemical, chemical or other means.
In some embodiments, a fragmentation site can include a nucleotide
or nucleotide sequence that may be fragmented by various means. For
example, a fragmentation site may comprise a restriction
endonuclease site; at least one ribonucleotide cleavable with an
RNAse; nucleotide analogues cleavable in the presence of certain
chemical agent; a diol linkage cleavable by treatment with
periodate; a disulphide group cleavable with a chemical reducing
agent; a cleavable moiety that may be subject to photochemical
cleavage; and a peptide cleavable by a peptidase enzyme or other
suitable means. See e.g., U.S. Patent Application Pub. No.
2012/0208705, U.S. Patent Application Pub. No. 2012/0208724 and
Int. Patent Application Pub. No. WO 2012/061832, each of which is
incorporated by reference in its entirety.
Primer Sites
[0119] In some embodiments, a transposon sequence can include a
"sequencing adaptor" or "sequencing adaptor site", that is to say a
region that comprises one or more sites that can hybridize to a
primer. In some embodiments, a transposon sequence can include at
least a first primer site useful for amplification, sequencing, and
the like. In some embodiments comprising looped transposomes, a
linker can include a sequencing adaptor. In more embodiments
comprising looped transposomes, a linker comprises at least a first
primer site and a second primer site. The orientation of the primer
sites in such embodiments can be such that a primer hybridizing to
the first primer site and a primer hybridizing to the second primer
site are in the same orientation, or in different orientations.
[0120] In some embodiments, a linker can include a first primer
site, a second primer site having a non-amplifiable site disposed
therebetween. The non-amplifiable site is useful to block extension
of a polynucleotide strand between the first and second primer
sites, wherein the polynucleotide strand hybridizes to one of the
primer sites. The non-amplifiable site can also be useful to
prevent concatamers. Examples of non-amplifiable sites include a
nucleotide analogue, non-nucleotide chemical moiety, amino-acid,
peptide, and polypeptide. In some embodiments, a non-amplifiable
site comprises a nucleotide analogue that does not significantly
base-pair with A, C, G or T. Some embodiments include a linker
comprising a first primer site, a second primer site having a
fragmentation site disposed therebetween. Other embodiments can use
a forked or Y-shaped adapter design useful for directional
sequencing, as described in U.S. Pat. No. 7,741,463, the disclosure
of which is incorporated herein by reference in its entirety.
Affinity Tags
[0121] In some embodiments, a transposon sequence or transposase
can include an affinity tag. In some embodiments comprising looped
transposomes a linker can comprise an affinity tag. Affinity tags
can be useful for a variety of applications, for example the bulk
separation of target nucleic acids hybridized to hybridization
tags. Additional application include, but are not limited to, using
affinity tags for purifying transposase/transposon complexes and
transposon inserted target DNA, for example. As used herein, the
term "affinity tag" and grammatical equivalents can refer to a
component of a multi-component complex, wherein the components of
the multi-component complex specifically interact with or bind to
each other. For example an affinity tag can include biotin or
poly-His that can bind streptavidin or nickel, respectively. Other
examples of multiple-component affinity tag complexes are listed,
for example, U.S. Patent Application Pub. No. 2012/0208705, U.S.
Patent Application Pub. No. 2012/0208724 and Int. Patent
Application Pub. No. WO 2012/061832, each of which is incorporated
by reference in its entirety.
Reporter Moieties
[0122] In some embodiments of the compositions and methods
described herein, a transposon sequence or transposase can include
a reporter moiety. In some embodiments comprising looped
transposomes a linker can comprise a reporter moiety. As used
herein, the term "reporter moiety" and grammatical equivalents can
refer to any identifiable tag, label, or group. The skilled artisan
will appreciate that many different species of reporter moieties
can be used with the methods and compositions described herein,
either individually or in combination with one or more different
reporter moieties. In certain embodiments, a reporter moiety can
emit a signal. Examples of a signal includes, but is not limited
to, a fluorescent, a chemiluminescent, a bioluminescent, a
phosphorescent, a radioactive, a calorimetric, an ion activity, an
electronic or an electrochemiluminescent signals. Example reporter
moieties are listed, for example, U.S. Patent Application Pub. No.
2012/0208705, U.S. Patent Application Pub. No. 2012/0208724 and
Int. Patent Application Pub. No. WO 2012/061832, each of which is
incorporated by reference in its entirety.
Certain Methods of Making Transposon Sequences
[0123] The transposon sequences provided herein can be prepared by
a variety of methods. Exemplary methods include direct synthesis ,
hairpin extension methods, and PCR. In some embodiments, transposon
sequences may be prepared by direct synthesis. For example, a
transposon sequence comprising a nucleic acid may be prepared by
methods comprising chemical synthesis. Such methods are well known
in the art, e.g., solid phase synthesis using phosphoramidite
precursors such as those derived from protected
2'-deoxynucleosides, ribonucleosides, or nucleoside analogues.
Example methods of preparing transposon sequencing can be found in,
for example, U.S. Patent Application Pub. No. 2012/0208705, U.S.
Patent Application Pub. No. 2012/0208724 and Int. Patent
Application Pub. No. WO 2012/061832, each of which is incorporated
by reference in its entirety.
[0124] In some embodiments comprising looped transposomes
transposon sequences comprising a single stranded linker can be
prepared. In some embodiments, the linker couples the transposon
sequences of a transposome such that a transposon sequence
comprising a first transposase recognition sequence is coupled to a
second transposon sequence comprising a second transposase
recognition sequence in a 5' to 3' orientation. In some
embodiments, the linker couples a transposon sequence comprising a
first transposase recognition sequence to a second transposon
sequence comprising a second transposase recognition sequence in a
5' to 5' orientation or in a 3' to 3' orientation. Coupling
transposon sequences of a transposome in either a 5' to 5'
orientation or in a 3' to 3' orientation can be advantageous to
prevent transposase recognition elements, in particular mosaic
elements (ME or M), from interacting with one another. For example,
coupled transposon sequences can be prepared by preparing
transposon sequences comprising either an aldehyde group or
oxyamine group. The aldehyde and oxyamine groups can interact to
form a covalent bond thus coupling the transposon sequences.
[0125] In some embodiments, transposomes comprising complementary
sequences can be prepared. FIG. 2 illustrates an embodiment in
which a transposase is loaded with transposon sequences comprising
complementary tails. The tails hybridize to form a linked
transposon sequence. Hybridization may occur in dilute conditions
to decrease the likelihood of hybridization between
transposomes.
Target Nucleic Acids
[0126] A target nucleic acid can include any nucleic acid of
interest. Target nucleic acids can include DNA, RNA, peptide
nucleic acid, morpholino nucleic acid, locked nucleic acid, glycol
nucleic acid, threose nucleic acid, mixed samples of nucleic acids,
polyploidy DNA (i.e., plant DNA), mixtures thereof, and hybrids
thereof. In a preferred embodiment, genomic DNA or amplified copies
thereof are used as the target nucleic acid. In another preferred
embodiment, cDNA, mitochondrial DNA or chloroplast DNA is used.
[0127] A target nucleic acid can comprise any nucleotide sequence.
In some embodiments, the target nucleic acid comprises homopolymer
sequences. A target nucleic acid can also include repeat sequences.
Repeat sequences can be any of a variety of lengths including, for
example, 2, 5, 10, 20, 30, 40, 50, 100, 250, 500 or 1000
nucleotides or more. Repeat sequences can be repeated, either
contiguously or non-contiguously, any of a variety of times
including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times
or more.
[0128] Some embodiments described herein can utilize a single
target nucleic acid. Other embodiments can utilize a plurality of
target nucleic acids. In such embodiments, a plurality of target
nucleic acids can include a plurality of the same target nucleic
acids, a plurality of different target nucleic acids where some
target nucleic acids are the same, or a plurality of target nucleic
acids where all target nucleic acids are different. Embodiments
that utilize a plurality of target nucleic acids can be carried out
in multiplex formats so that reagents are delivered simultaneously
to the target nucleic acids, for example, in one or more chambers
or on an array surface. In some embodiments, the plurality of
target nucleic acids can include substantially all of a particular
organism's genome. The plurality of target nucleic acids can
include at least a portion of a particular organism's genome
including, for example, at least about 1%, 5%, 10%, 25%, 50%, 75%,
80%, 85%, 90%, 95%, or 99% of the genome. In particular embodiments
the portion can have an upper limit that is at most about 1%, 5%,
10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the genome
[0129] Target nucleic acids can be obtained from any source. For
example, target nucleic acids may be prepared from nucleic acid
molecules 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, or
organisms. Cells that may be used as sources of target nucleic acid
molecules may be prokaryotic (bacterial cells, for example,
Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus,
Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema,
Mycoplasma, Borrelia, Legionella, Pseudomonas, Mycobacterium,
Helicobacter, Erwinia, Agrobacterium, Rhizobium, and Streptomyces
genera); archeaon, such as crenarchaeota, nanoarchaeota or
euryarchaeotia; or eukaryotic such as fungi, (for example, yeasts),
plants, protozoans and other parasites, and animals (including
insects (for example, Drosophila spp.), nematodes (e.g.,
Caenorhabditis elegans), and mammals (for example, rat, mouse,
monkey, non-human primate and human). Target nucleic acids and
template nucleic acids can be enriched for certain sequences of
interest using various methods well known in the art. Examples of
such methods are provided in Int. Pub. No. WO/2012/108864, which is
incorporated herein by reference in its entirety. In some
embodiments, nucleic acids may be further enriched during methods
of preparing template libraries. For example, nucleic acids may be
enriched for certain sequences, before insertion of transposomes
after insertion of transposomes and/or after amplification of
nucleic acids.
[0130] In addition, in some embodiments, target nucleic acids
and/or template nucleic acids can be highly purified, for example,
nucleic acids can be at least about 70%, 80%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% free from contaminants before use with the
methods provided herein. In some embodiments, it is beneficial to
use methods known in the art that maintain the quality and size of
the target nucleic acid, for example isolation and/or direct
transposition of target DNA may be performed using agarose plugs.
Transposition can also be performed directly in cells, with
population of cells, lysates, and non-purified DNA.
Certain Methods of Preparing Template Nucleic Acids
[0131] Some embodiments include methods of preparing template
nucleic acids. As used herein, "template nucleic acid" can refer to
a substrate for obtaining sequence information. In some
embodiments, a template nucleic acid can include a target nucleic
acid, a fragment thereof, or any copy thereof comprising at least
one transposon sequence, a fragment thereof, or any copy thereof.
In some embodiments, a template nucleic acid can include a target
nucleic acid comprising a sequencing adaptor, such as a sequencing
primer site.
[0132] Some methods of preparing template nucleic acids include
inserting a transposon sequence into a target nucleic acid, thereby
preparing a template nucleic acid. Some methods of insertion
include contacting a transposon sequence provided herein with a
target nucleic acid in the presence of an enzyme, such as a
transposase or integrase, under conditions sufficient for the
integration of the transposon sequence or sequences into the target
nucleic acid.
[0133] In some embodiments, insertion of transposon sequences into
a target nucleic acid can be non-random. In some embodiments,
transposon sequences can be contacted with target nucleic acids
comprising proteins that inhibit integration at certain sites. For
example, transposon sequences can be inhibited from integrating
into genomic DNA comprising proteins, genomic DNA comprising
chromatin, genomic DNA comprising nucleosomes, or genomic DNA
comprising histones. In some embodiments, transposon sequences can
be associated with affinity tags in order to integrate the
transposon sequence at a particular sequence in a target nucleic
acid. For example, a transposon sequence may be associated with a
protein that targets specific nucleic acid sequences, e.g.,
histones, chromatin-binding proteins, transcription factors,
initiation factors, etc., and antibodies or antibody fragments that
bind to particular sequence-specific nucleic-acid-binding proteins.
In an exemplary embodiment, a transposon sequence is associated
with an affinity tag, such as biotin; the affinity tag can be
associated with a nucleic-acid-binding protein.
[0134] It will be understood that during integration of some
transposon sequences into a target nucleic acid, several
consecutive nucleotides of the target nucleic acid at the
integration site are duplicated in the integrated product. Thus the
integrated product can include a duplicated sequence at each end of
the integrated sequence in the target nucleic acid. As used herein,
the term "host tag" or "g-tag" can refer to a target nucleic acid
sequence that is duplicated at each end of an integrated transposon
sequence. Single-stranded portions of nucleic acids that may be
generated by the insertion of transposon sequences can be repaired
by a variety of methods well known in the art, for example by using
ligases, oligonucleotides and/or polymerases.
[0135] In some embodiments, a plurality of the transposon sequences
provided herein is inserted into a target nucleic acid. Some
embodiments include selecting conditions sufficient to achieve
integration of a plurality of transposon sequences into a target
nucleic acid such that the average distance between each integrated
transposon sequence comprises a certain number of consecutive
nucleotides in the target nucleic acid.
[0136] Some embodiments include selecting conditions sufficient to
achieve insertion of a transposon sequence or sequences into a
target nucleic acid, but not into another transposon sequence or
sequences. A variety of methods can be used to reduce the
likelihood that a transposon sequence inserts into another
transposon sequence. Examples of such methods useful with the
embodiments provided herein can be found in for example, U.S.
Patent Application Pub. No. 2012/0208705, U.S. Patent Application
Pub. No. 2012/0208724 and Int. Patent Application Pub. No. WO
2012/061832, each of which is incorporated by reference in its
entirety.
[0137] In some embodiments, conditions may be selected so that the
average distance in a target nucleic acid between integrated
transposon sequences is at least about 5, 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, or more consecutive nucleotides. In some
embodiments, the average distance in a target nucleic acid between
integrated transposon sequences is at least about 100, 200, 300,
400, 500, 600, 700, 800, 900, 1000, or more consecutive
nucleotides. In some embodiments, the average distance in a target
nucleic acid between integrated transposon sequences is at least
about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 90 kb, 100
kb, or more consecutive nucleotides. In some embodiments, the
average distance in a target nucleic acid between integrated
transposon sequences is at least about 100 kb, 200 kb, 300 kb, 400
kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, or more
consecutive nucleotides. As will be understood, some conditions
that may be selected include contacting a target nucleic acid with
a certain number of transposon sequences.
[0138] Some embodiments of the methods described herein include
selecting conditions sufficient to achieve at least a portion of
transposon sequences integrated into a target nucleic acid that are
different. In preferred embodiments of the methods and compositions
described herein, each transposon sequence integrated into a target
nucleic acid is different. Some conditions that may be selected to
achieve a certain portion of transposon sequences integrated into
target sequences that are different include selecting the degree of
diversity of the population of transposon sequences. As will be
understood, the diversity of transposon sequences arises in part
due to the diversity of the barcodes of such transposon sequences.
Accordingly, some embodiments include providing a population of
transposon sequences in which at least a portion of the barcodes
are different. In some embodiments, at least about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of barcodes in
a population of transposon sequences are different. In some
embodiments, at least a portion of the transposon sequences
integrated into a target nucleic acid are the same.
[0139] Some embodiments of preparing a template nucleic acid can
include copying the sequences comprising the target nucleic acid.
For example, some embodiments include hybridizing a primer to a
primer site of a transposon sequence integrated into the target
nucleic acid. In some such embodiments, the primer can be
hybridized to the primer site and extended. The copied sequences
can include at least one barcode sequence and at least a portion of
the target nucleic acid. In some embodiments, the copied sequences
can include a first barcode sequence, a second barcode sequence,
and at least a portion of a target nucleic acid disposed
therebetween. In some embodiments, at least one copied nucleic acid
can include at least a first barcode sequence of a first copied
nucleic acid that can be identified or designated to be paired with
a second barcode sequence of a second copied nucleic acid. In some
embodiments, the primer can include a sequencing primer. In some
embodiments sequencing data is obtained using the sequencing
primer. In more embodiments, adaptors comprising primer sites can
be ligated to each end of a nucleic acid, and the nucleic amplified
from such primer sites.
[0140] Some embodiments of preparing a template nucleic acid can
include amplifying sequences comprising at least a portion of one
or more transposon sequences and at least a portion of a target
nucleic acid. In some embodiments, at least a portion of a target
nucleic acid can be amplified using primers that hybridize to
primer sites of integrated transposon sequences integrated into a
target nucleic acid. In some such embodiments, an amplified nucleic
acid can include a first barcode sequence, and second barcode
sequence having at least a portion of the target nucleic acid
disposed therebetween. In some embodiments, at least one amplified
nucleic acid can include at least a first barcode sequence of a
first amplified nucleic acid that can be identified to be paired
with a second barcode sequence of a second amplified sequence.
[0141] Some methods of preparing template nucleic acids include
inserting transposon sequences comprising single-stranded linkers.
FIG. 3 illustrates an example in which transposon sequences
(ME-P1-linker-P2-ME; mosaic end-primer site 1-linker-primer site
2-mosaic end) are inserted into a target nucleic acid. The target
nucleic acid having the inserted transposon/linker sequences can be
extended and amplified.
[0142] In one embodiment of the compositions and methods described
herein, transposomes are used that have symmetrical transposable
end sequences to produce an end-tagged target nucleic acid fragment
(tagmented fragment or tagment). Each tagmented fragment therefore
contains identical ends, lacking directionality. A single primer
PCR, using the transposon end sequences, can then be employed to
amplify the template copy number from 2n to 2n*2.sup.x where x
corresponds to the number of PCR cycles. In a subsequent step, PCR
with primers can add additional sequences, such as sequencing
adapter sequences.
[0143] In some embodiments, it can be advantageous for each
template nucleic acid to incorporate at least one universal primer
site. For example, a template nucleic, acid can include first end
sequences that comprise a first universal primer site, and second
end sequences that comprise a second universal primer site.
Universal primer sites can have various applications, such as use
in amplifying, sequencing, and/or identifying one or more template
nucleic acids. The first and second universal primer sites can be
the same, substantially similar, similar, or different. Universal
primer sites can be introduced into nucleic acids by various
methods well known in the art, for example, ligation of primer
sites to nucleic acids, amplification of nucleic acids using tailed
primers, and insertion of a transposon sequence comprising a
universal primer site.
Targeted Insertion
[0144] In some embodiments of the methods and compositions provided
herein, transposon sequences may be inserted at particular targeted
sequences of a target nucleic acid. Transposition into dsDNA can be
more efficient than into ssDNA targets. In some embodiments, dsDNA
is denatured into ssDNA and annealed with oligonucleotide probes
(20-200 bases). These probes create sites of dsDNA that can be
efficiently used as integration sites with transposomes provided
herein. In some embodiments, dsDNA can be targeted using D-loop
formation with recA-coated oligo probes, and subsequent triplex
formation. In some such embodiments, the replication fork structure
is the preferred substrate for transposomes comprising Tn4430
transposase. In more embodiments, regions of interest in dsDNA can
be targeted using sequence-specific DNA binding proteins such as
zinc-finger complexes, and other affinity ligands to specific DNA
regions.
[0145] In some embodiments, transposomes comprising a transposase
having a preferred substrate of mismatched positions in a target
nucleic acid may be used to target insertion into the target
nucleic acid. For example, some MuA transposases, such as HYPERMU
(Epicenter), have a preference for mismatched targets. In some such
embodiments, oligonucleotide probes comprising a mismatch are
annealed to a single-stranded target nucleic acid. Transposomes
comprising MuA transposases, such as HYPERMU, can be used to target
the mismatched sequences of the target nucleic acid.
Fragmenting Template Nucleic Acids
[0146] Some embodiments of preparing a template nucleic acid can
include fragmenting a target nucleic acid. In some embodiments,
insertion of transposomes comprising non-contiguous transposon
sequences can result in fragmentation of a target nucleic acid. In
some embodiments comprising looped transposomes a target nucleic
acid comprising transposon sequences can be fragmented at the
fragmentation sites of the transposon sequences. Further examples
of method useful to fragment target nucleic acids useful with the
embodiments provided herein can be found in for example, U.S.
Patent Application Pub. No. 2012/0208705, U.S. Patent Application
Pub. No. 2012/0208724 and Int. Patent Application Pub. No. WO
2012/061832, each of which is incorporated by reference in its
entirety.
Tagging Single Molecules
[0147] The present invention provides methods for tagging molecules
so that individual molecules can be tracked and identified. The
bulk data can then be deconvoluted and converted back to the
individual molecule. The ability to distinguish individual
molecules and relate the information back to the molecule of origin
is especially important when processes from original molecule to
final product change the (stoichiometric) representation of the
original population. For example, amplification leads to
duplication (e.g., PCR duplicates or biased amplification) that can
skew the original representation. This can alter the methylation
state call, copy number, allelic ratio due to non-uniform
amplification and/or amplification bias. By identifying individual
molecules, code-tagging distinguishes between identical molecules
after processing. As such, duplications, and amplification bias can
be filtered out, allowing accurate determination of the original
representation of a molecule or population of molecules.
[0148] An advantage of uniquely tagging single molecules is that
identical molecules in the original pool become uniquely identified
by virtue of their tagging. In further downstream analyses, these
uniquely tagged molecules can now be distinguished. This technique
can be exploited in assay schemes in which amplification is
employed. For example, amplification is known to distort the
original representation of a mixed population of molecules. If
unique tagging were not employed, the original representation (such
as copy number or allelic ratio) would need to account for the
biases (known or unknown) for each molecule in the representation.
With unique tagging, the representation can accurately be
determined by removing duplicates and counting the original
representation of molecules, each having a unique tag. Thus, cDNAs
can be amplified and sequenced, without fear of bias because the
data can be filtered so that only authentic sequences or sequences
of interest are selected for further analysis. Accurate reads can
be constructed by taking the consensus across many reads with the
same barcode.
[0149] In some embodiments of the compositions and methods
described herein, it is preferred to tag the original population in
the early stages of the assay, although tagging can occur at later
stages if the earlier steps do not introduce bias or are not
important. In any of these applications, the complexity of the
barcode sequences should be larger than the number of individual
molecules to be tagged. This ensures that different target
molecules receive different and unique tags. As such, a pool of
random oligonucleotides of a certain length (e.g., 5, 10, 20, 30,
40, 50, 100 or 200 nucleotides in length) is desirable. A random
pool of tags represents a large complexity of tags with code space
4.sup.n where n is the number of nucleotides. Additional codes
(whether designed or random) can be incorporated at different
stages to serve as a further check, such as a parity check for
error correction.
[0150] In one embodiment of the compositions and methods described
herein, individual molecules (such as target DNA) are attached to
unique labels, such as unique oligo sequences and/or barcodes.
Attachment of the labels can occur through ligation, coupling
chemistry, adsorption, insertion of transposon sequences, etc.
Other means include amplification (such as by PCR, RCA or LCR),
copying (such as addition by a polymerase), and non-covalent
interactions.
[0151] Specific methods comprise including barcodes (e.g., designed
or random sequences) to PCR primers so that each template will
receive an individual code within the code space, thereby yielding
unique amplicons that can be discriminated from other amplicons.
This concept can be applied to any method that uses polymerase
amplification, such as GoldenGate assays as disclosed in U.S. Pat.
Nos. 7,582,420, 7,955,794, and 8,003,354, each of which is
incorporated by reference in its entirety. Code-tagged target
sequences can be circularized and amplified by methods such as
rolling-circle amplification to yield code-tagged amplicons.
Similarly, the code can also be added to RNA
Methods of Analyzing Template Nucleic Acids
[0152] Some embodiments of the technology described herein include
methods of analyzing template nucleic acids. In such embodiments,
sequencing information can be obtained from template nucleic acids
and this information can be used to generate a sequence
representation of one or more target nucleic acids.
[0153] In some embodiments of the sequencing methods described
herein, a linked read strategy may be used. A linked read strategy
can include identifying sequencing data that links at least two
sequencing reads. For example, a first sequencing read may contain
a first marker, and a second sequencing read may contain a second
marker. The first and second markers can identify the sequencing
data from each sequencing read to be adjacent in a sequence
representation of the target nucleic acid. In some embodiments of
the compositions and methods described herein, markers can comprise
a first barcode sequence and a second barcode sequence in which the
first barcode sequence can be paired with the second barcode
sequence. In other embodiments, markers can comprise a first host
tag and a second host tag. In more embodiments, markers can
comprise a first barcode sequence with a first host tag, and a
second barcode sequence with a second host tag
[0154] An exemplary embodiment of a method for sequencing a
template nucleic acid can comprise the following steps: (a)
sequence the first barcode sequence using a sequencing primer
hybridizing to the first primer site; and (b) sequence the second
barcode sequence using a sequencing primer hybridizing to the
second primer. The result is two sequence reads that help link the
template nucleic acid to its genomic neighbors. Given long enough
reads, and short enough library fragments, these two reads can be
merged informatically to make one long read that covers the entire
fragment. Using the barcode sequence reads and the 9 nucleotide
duplicated sequence present from the insertion, reads can now be
linked to their genomic neighbors to form much longer "linked
reads" in silico.
[0155] As will be understood, a library comprising template nucleic
acids can include duplicate nucleic acid fragments. Sequencing
duplicate nucleic acid fragments is advantageous in methods that
include creating a consensus sequence for duplicate fragments. Such
methods can increase the accuracy for providing a consensus
sequence for a template nucleic acid and/or library of template
nucleic acids.
[0156] In some embodiments of the sequencing technology described
herein, sequence analysis is performed in real time. For example,
real time sequencing can be performed by simultaneously acquiring
and analyzing sequencing data. In some embodiments, a sequencing
process to obtain sequencing data can be terminated at various
points, including after at least a portion of a target nucleic acid
sequence data is obtained or before the entire nucleic acid read is
sequenced. Exemplary methods, systems, and further embodiments are
provided in International Patent Publication No. WO 2010/062913,
the disclosure of which is incorporated herein by reference in its
entirety.
[0157] In an exemplary embodiment of a method for assembling short
sequencing reads using a linked read strategy, transposon sequences
comprising barcodes are inserted into genomic DNA, a library is
prepared and sequencing data is obtained for the library of
template nucleic acids. Blocks of templates can be assembled by
identifying paired barcodes and then larger contigs are assembled.
In one embodiment, the assembled reads can be further assembled
into larger contigs through code pairing using overlapping
reads.
[0158] Some embodiments of the sequencing technology described
herein include error detection and correction features. Examples of
errors can include errors in base calls during a sequencing
process, and errors in assembling fragments into larger contigs. As
would be understood, error detection can include detecting the
presence or likelihood of errors in a data set, and as such,
detecting the location of an error or number of errors may not be
required. For error correction, information regarding the location
of an error and/or the number of errors in a data set is useful.
Methods for error correction are well known in the art. Examples
include the use of hamming distances, and the use of a checksum
algorithm (See, e.g., U.S. Patent Application Publication No.
2010/0323348; U.S. Pat. Nos. 7,574,305; and 6,654,696, the
disclosures of which are incorporated herein by reference in their
entireties).
Nested Libraries
[0159] An alternative method involves the junction tagging methods
above and preparation of nested sequencing libraries. The nested
sub-libraries are created from code-tagged DNA fragments. This can
allow less frequent transposon tagging across the genome. It can
also create a larger diversity of (nested) sequencing reads. These
factors can lead to improved coverage and accuracy.
[0160] Sub-sampling and whole genome amplification can create many
copies of a certain population of starting molecules. DNA fragments
are then generated by transposon-specific fragmentation, where each
fragment receives a code that allows one to link the fragment back
to the original neighbor having a matching code (whether identical,
complementary or otherwise informatically linked). The tagged
fragments are fragmented at least a second time by random methods
or sequence-specific methods, such as enzymatic digestion, random
shearing, transposon-based shearing or other methods, thereby
creating sub-libraries of the code-tagged DNA fragments. In a
useful variation of the previously-described method, code-tagged
fragments can be preferentially isolated by using transposons that
contain a biotin or other affinity functionality for downstream
enrichment purposes. Subsequent library preparation converts the
nested DNA fragments into sequencing templates. Paired-end
sequencing results in determination of the sequence of the code-tag
of the DNA fragments and of the target DNA. Since nested libraries
for the same code-tag are created, long DNA fragments can be
sequenced with short reads.
Sequencing Methods
[0161] The methods and composition described herein can be used in
conjunction with a variety of sequencing techniques. In some
embodiments, the process to determine the nucleotide sequence of a
target nucleic acid can be an automated process.
[0162] Some embodiments of the sequencing methods described herein
include sequencing by synthesis (SBS) technologies, for example,
pyrosequencing techniques. Pyrosequencing detects the release of
inorganic pyrophosphate (PP.sub.i) as particular nucleotides are
incorporated into the nascent strand (Ronaghi et al., Analytical
Biochemistry 242(1): 84-9 (1996); Ronaghi, M. Genome Res.
11(1):3-11 (2001); Ronaghi et al., Science 281(5375):363 (1998);
U.S. Pat. Nos. 6,210,891; 6,258,568 and 6,274,320, each of which is
incorporated by reference in its entirety).
[0163] In another example type of SBS, cycle sequencing is
accomplished by stepwise addition of reversible terminator
nucleotides containing, for example, a cleavable or photobleachable
dye label as described, for example, in U.S. Pat. Nos. 7,427,67,
7,414,1163 and 7,057,026, each of which is incorporated by
reference in its entirety. This approach, which is being
commercialized by Illumina Inc., is also described in International
Patent Application Publication Nos. WO 91/06678 and WO 07/123744,
each of which is incorporated by reference in its entirety. The
availability of fluorescently-labeled terminators, in which both
the termination can be reversed and the fluorescent label cleaved,
facilitates efficient cyclic reversible termination (CRT)
sequencing. Polymerases can also be co-engineered to efficiently
incorporate and extend from these modified nucleotides.
[0164] Additional exemplary SBS systems and methods which can be
utilized with the methods and compositions described herein are
described in U.S. Patent Application Publication No. 2007/0166705,
U.S. Patent Application Publication No. 2006/0188901, U.S. Pat. No.
7,057,026, U.S. Patent Application Publication No. 2006/0240439,
U.S. Patent Application Publication No. 2006/0281109, PCT
Publication No. WO 05/065814, U.S. Patent Application Publication
No. 2005/0100900, PCT Publication No. WO 06/064199 and PCT
Publication No. WO 07/010251, each of which is incorporated by
reference in its entirety.
[0165] Some embodiments of the sequencing technology described
herein can utilize sequencing by ligation techniques. Such
techniques utilize DNA ligase to incorporate nucleotides and
identify the incorporation of such nucleotides. Exemplary SBS
systems and methods which can be utilized with the compositions and
methods described herein are described in U.S. Pat. Nos. 6,969,488,
6,172,218, and 6,306,597, each of which is incorporated by
reference in its entirety.
[0166] Some embodiments of the sequencing technology described
herein can include techniques such as next-next technologies. One
example can include nanopore sequencing techniques (Deamer, D. W.
& Akeson, M. "Nanopores and nucleic acids: prospects for
ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000);
Deamer, D. and D. Branton, "Characterization of nucleic acids by
nanopore analysis". Acc. Chem. Res. 35:817-825 (2002); Li et al.,
"DNA molecules and configurations in a solid-state nanopore
microscope" Nat. Mater. 2:611-615 (2003), each of which is
incorporated by reference in its entirety). In such embodiments,
the target nucleic acid passes through a nanopore. The nanopore can
be a synthetic pore or biological membrane protein, such as
.alpha.-hemolysin. As the target nucleic acid passes through the
nanopore, each base-pair can be identified by measuring
fluctuations in the electrical conductance of the pore. (U.S. Pat.
No. 7,001,792; Soni & Meller, "A. Progress toward ultrafast DNA
sequencing using solid-state nanopores," Clin. Chem. 53, 1996-2001
(2007); Healy, K. "Nanopore-based single-molecule DNA analysis,"
Nanomed. 2:459-481 (2007); Cockroft et al., "A single-molecule
nanopore device detects DNA polymerase activity with
single-nucleotide resolution." J. Am. Chem. Soc. 130:818-820
(2008), each of which is incorporated by reference in its
entirety). In some such embodiments, nanopore sequencing techniques
can be useful to confirm sequence information generated by the
methods described herein.
[0167] Some embodiments of the sequencing technology described
herein can utilize methods involving the real-time monitoring of
DNA polymerase activity. Nucleotide incorporations can be detected
through fluorescence resonance energy transfer (FRET) interactions
between a fluorophore-hearing polymerase and
.gamma.-phosphate-labeled nucleotides as described, for example, in
U.S. Pat. Nos. 7,329,492 and 7,211,414 or nucleotide incorporations
can be detected with zero-mode waveguides as described, for
example, in U.S. Pat. No. 7,315,019 and using fluorescent
nucleotide analogs and engineered polymerases as described, for
example, in U.S. Pat. No. 7,405,281 and U.S. Patent Application
Publication No. 2008/0108082, each of which is incorporated by
reference in its entirety. The illumination can be restricted to a
zeptoliter-scale volume around a surface-tethered polymerase such
that incorporation of fluorescently labeled nucleotides can be
observed with low background (Levene, M. J. et al. "Zero-mode
waveguides for single-molecule analysis at high concentrations."
Science 299, 682-686 (2003); Lundquist, P. M. et al. "Parallel
confocal detection of single molecules in real time." Opt. Lett.
33, 1026-1028 (2008); Korlach, J, et al. "Selective aluminum
passivation for targeted immobilization of single DNA polymerase
molecules in zero-mode waveguide nanostructures," Proc. Natl. Acad.
Sci. USA 105, 1176-1181 (2008), each of which is incorporated by
reference in its entirety). In one example, single molecule,
real-time (SMRT) DNA sequencing technology provided by Pacific
Biosciences Inc. can be utilized with the methods described herein.
In some embodiments, a SMRT chip or the like may be utilized (e.g.,
U.S. Pat. Nos. 7,181,122, 7,302,146 and 7,313,308, each of which is
incorporated by reference in its entirety). A SMRT chip comprises a
plurality of zero-mode waveguides (ZMW). Each ZMW comprises a
cylindrical hole tens of nanometers in diameter perforating a thin
metal film supported by a transparent substrate. When the ZMW is
illuminated through the transparent substrate, attenuated light may
penetrate the lower 20-30 nm of each ZMW creating a detection
volume of about 1.times.10.sup.-21 L. Smaller detection volumes
increase the sensitivity of detecting fluorescent signals by
reducing the amount of background that can be observed.
[0168] SMRT chips and similar technology can be used in association
with nucleotide monomers fluorescently labeled on the terminal
phosphate of the nucleotide (Korlach J, et al., "Long, processive
enzymatic DNA synthesis using 100% dye-labeled terminal
phosphate-linked nucleotides." Nucleosides, Nucleotides and Nucleic
Acids, 27:1072-1083, 2008, which is incorporated by reference in
its entirety). The label is cleaved from the nucleotide monomer on
incorporation of the nucleotide into the polynucleotide.
Accordingly, the label is not incorporated into the polynucleotide,
increasing the signal: background ratio. Moreover, the need for
conditions to cleave a label from labeled nucleotide monomers is
reduced.
[0169] An additional example of a sequencing platform that may be
used in association with some of the embodiments described herein
is provided by Helicos Biosciences Corp. In some embodiments, TRUE
SINGLE MOLECULE SEQUENCING can be utilized (Harris T. D. et al.,
"Single Molecule DNA Sequencing of a viral Genome" Science
320:106-109 (2008), which is incorporated by reference in its
entirety). In one embodiment, a library of target nucleic acids can
be prepared by the addition of a 3' poly(A) tail to each target
nucleic acid. The poly(A) tail hybridizes to poly(T)
oligonucleotides anchored on a glass cover slip. The poly(T)
oligonucleotide can be used as a primer for the extension of a
polynucleotide complementary to the target nucleic acid. In one
embodiment, fluorescently-labeled nucleotide monomers, namely, A,
C, G, or T, are delivered one at a time to the target nucleic acid
in the presence DNA polymerase. Incorporation of a labeled
nucleotide into the polynucleotide complementary to the target
nucleic acid is detected, and the position of the fluorescent
signal on the glass cover slip indicates the molecule that has been
extended. The fluorescent label is removed before the next
nucleotide is added to continue the sequencing cycle. Tracking
nucleotide incorporation in each polynucleotide strand can provide
sequence information for each individual target nucleic acid.
[0170] An additional example of a sequencing platform that can be
used in association with the methods described herein is provided
by Complete Genomics Inc. Libraries of target nucleic acids can be
prepared where target nucleic acid sequences are interspersed
approximately every 20 bp with adaptor sequences. The target
nucleic acids can be amplified using rolling circle replication,
and the amplified target nucleic acids can be used to prepare an
array of target nucleic acids. Methods of sequencing such arrays
include sequencing by ligation, in particular, sequencing by
combinatorial probe-anchor ligation (cPAL).
[0171] In some embodiments using cPAL, about 10 contiguous bases
adjacent to an adaptor may be determined. A pool of probes that
includes four distinct labels for each base (A, C, T, G) is used to
read the positions adjacent to each adaptor. A separate pool is
used to read each position. A pool of probes and an anchor specific
to a particular adaptor is delivered to the target nucleic acid in
the presence of ligase. The anchor hybridizes to the adaptor, and a
probe hybridizes to the target nucleic acid adjacent to the
adaptor. The anchor and probe are ligated to one another. The
hybridization is detected and the anchor-probe complex is removed.
A different anchor and pool of probes is then delivered to the
target nucleic acid in the presence of ligase.
[0172] The sequencing methods described herein can be
advantageously carried out in multiplex formats such that multiple
different target nucleic acids are manipulated simultaneously. In
particular embodiments, different target nucleic acids can be
treated in a common reaction vessel or on a surface of a particular
substrate. This allows convenient delivery of sequencing reagents,
removal of unreacted reagents and detection of incorporation events
in a multiplex manner. In embodiments using surface-bound target
nucleic acids, the target nucleic acids can be in an array format.
In an array format, the target nucleic acids can be typically
coupled to a surface in a spatially distinguishable manner. For
example, the target nucleic acids can be bound by direct covalent
attachment, attachment to a bead or other particle or associated
with a polymerase or other molecule that is attached to the
surface. The array can include a single copy of a target nucleic
acid at each site (also referred to as a feature) or multiple
copies having the same sequence can be present at each site or
feature. Multiple copies can be produced by amplification methods
such as, bridge amplification or emulsion PCR as described in
further detail herein.
[0173] The methods set forth herein can use arrays having features
at any of a variety of densities including, for example, at least
about 10 features/cm.sup.2, 100 features/cm.sup.2, 500
features/cm.sup.2, 1,000 features/cm.sup.2, 5,000
features/cm.sup.2, 10,000 features/cm.sup.2, 50,000
features/cm.sup.2, 100,000 features/cm.sup.2, 1,000,000
features/cm.sup.2, 5,000,000 features/cm.sup.2, 10.sup.7
features/cm.sup.2, 5.times.10.sup.7 features/cm.sup.2, 10.sup.8
features/cm.sup.2, 5.times.10.sup.8 features/cm.sup.2, 10.sup.9
features/cm.sup.2, 5.times.10.sup.9 features/cm.sup.2, or
higher.
Surfaces
[0174] In some embodiments, the nucleic acid template provided
herein can he attached to a solid support ("substrate"). Substrates
can be two-or three-dimensional and can comprise a planar surface
(e.g., a glass slide) or can be shaped. A substrate can include
glass (e.g., controlled pore glass (CPG)), quartz, plastic (such as
polystyrene (low cross-linked and high cross-linked polystyrene),
polycarbonate, polypropylene and poly(rnethylmethacrylate)),
acrylic copolymer, polyamide, silicon, metal (e.g.,
alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran,
gel matrix (e.g., silica gel), polyacrolein, or composites.
[0175] Suitable three-dimensional substrates include, for example,
spheres, microparticles, beads, membranes, slides, plates,
micromachined chips, tubes (e.g., capillary tubes), microwells,
microfluiclic devices, channels, filters, or any other structure
suitable for anchoring a nucleic acid. Substrates can include
planar arrays or matrices capable of having regions that include
populations of template nucleic acids or primers. Examples include
nucleoside-derivatized CPG and polystyrene slides; derivatized
magnetic slides; polystyrene grafted with polyethylene glycol, and
the like. Various methods well known in the art can be used to
attach, anchor or immobilize nucleic acids to the surface of the
substrate.
Methods for Reducing Error Rates in Sequencing Data
[0176] Some embodiments of the methods and compositions provided
herein include reducing the error rates in sequencing data. In some
such embodiments, the sense and antisense strands of a
double-stranded target nucleic acid are each associated with a
different barcode. Each strand is amplified, sequence information
is obtained from multiple copies of the amplified strands, and a
consensus sequence representation of the target nucleic acid is
generated from the redundant sequence information. Thus, sequence
information can originate and be identified from each strand.
Accordingly, sequence errors can be identified and reduced where
sequence information originating from one strand is inconsistent
with sequence information from the other strand.
[0177] In some embodiments, the sense and antisense strands of a
target nucleic acid are associated with a different barcode. The
barcodes may be associated with the target nucleic acid by a
variety of methods including ligation of adaptors and insertion of
transposon sequences. In some such embodiments, a Y-adaptor may be
ligated to at least one end of a target nucleic acid. The Y-adaptor
can include a double-stranded sequence, and non-complementary
strands, each strand comprising a different barcode. The target
nucleic acid with ligated Y-adaptor can be amplified and sequenced
such that each barcode can be used to identify the original sense
or antisense strands. A similar method is described in Kinde I. et
al., (2011) PNAS 108:9530-9535, the disclosure of which is
incorporated herein by reference in its entirety. In some
embodiments, the sense and antisense strands of a target nucleic
acid are associated with a different barcode by inserting
transposon sequences provided herein. In some such embodiments, the
transposon sequences can comprise non-complementary barcodes.
[0178] Some embodiments of such methods include obtaining sequence
information from a strand of a target double-stranded nucleic acid
comprising (a) obtaining sequence data from a template nucleic acid
comprising a first sequencing adapter and a second sequencing
adapter having at least a portion of the double-stranded target
nucleic acid disposed therebetween, wherein: (i) the first
sequencing adapter comprises a double-stranded first barcode, a
single-stranded first primer site and a single-stranded second
primer site, wherein the first and second primer sites are
non-complementary, and (ii) the second sequencing adapter
comprising a double-stranded second barcode, a single-stranded
third primer site and a single-stranded fourth primer site, wherein
the third and fourth primer sites are non-complementary. In some
embodiments, the first primer site of the sense strand of the
template nucleic acid and the third primer site of the antisense
sense strand of the template nucleic acid comprise the same
sequence. In some embodiments, each barcode is different. In some
embodiments, the first sequencing adapter comprises a
single-stranded hairpin coupling the first primer site and second
primer site.
[0179] In another embodiment, each end of a target nucleic acid is
associated with an adaptor comprising a different barcode such that
extension products from the sense and antisense strand of a nucleic
acid can be distinguished from each other. In some embodiments,
primer site sequences and barcodes are selected such that extension
from a primer annealed to the sense strand yields products that can
be distinguished from products of extension from a primer annealed
to the antisense strand. In an example, the 3' sense primer site is
the same as the 3' antisense primer site, but different from both
the 5' sense and 5' antisense primer sites. Extension of primers
annealed to the 3' sense primer site and the 3' antisense primer
site would yield the following products from each strand:
[0180] Sense strand: (5') barcode 2-[target sequence]-barcode 1
(3')
[0181] Antisense strand: (5') barcode 1-[target sequence]-barcode 2
(3')
[0182] Thus, extension products from the sense and antisense strand
of a nucleic acid can be distinguished from each other. An
exemplary method is illustrated in Schmitt M. W., et al., PNAS
(2012) 109:14508-13, the disclosure of which is incorporated herein
by reference in its entirety. In some such methods, the barcodes
and primers sites may be associated with the target nucleic acid by
a variety of methods including ligation of adaptors and insertion
of transposon sequences. In some embodiments, transposon sequences
can be designed to provide adaptors with hairpins. Hairpins provide
the ability to maintain the physical contiguity of the sense and
antisense strands of a target nucleic acid. A template nucleic acid
can be prepared comprising hairpins using transposon sequences
comprising linkers described herein. Examples of linkers include
single-stranded nucleic acids.
[0183] Some embodiments of preparing a library of template nucleic
acids for obtaining sequence information from each strand of a
double-stranded target nucleic acid include (a) providing a
population of transposomes comprising a transposase and a first
transposon sequence comprising: (i) a first transposase recognition
site, a first primer site, and a first barcode, and (ii) a second
transposon sequence comprising a second transposase recognition
site, a second primer site, and a second barcode, wherein the first
transposon sequence is non-contiguous with the second transposon
sequence; and (b) contacting the transposomes with a
double-stranded nucleic acid under conditions such that said first
and second transposon sequences insert into the double-stranded
target nucleic acid, thereby preparing a library of template
nucleic acids for obtaining sequence information from each strand
of the double-stranded target nucleic acid. In some embodiments,
the population of transposomes further comprises transposomes
comprising a transposase and a transposon sequence comprising a
third transposase recognition site and a fourth transposase
recognition site having a barcode sequence disposed therebetween,
said barcode sequence comprising a third barcode and a fourth
barcode having a sequencing adapter disposed therebetween, said
sequencing adapter comprising a third primer site and a fourth
primer site having a linker disposed therebetween. In some
embodiments, the first primer site of the sense strand of the
template nucleic acid and the third primer site of the antisense
sense strand of the template nucleic acid comprise the same
sequence. Some embodiments also include a step (c) selecting for
template nucleic acids comprising transposon sequences wherein the
first transposon sequence is non-contiguous with the second
transposon sequence and transposon sequences comprising a linker.
In some embodiments, the linker comprises an affinity tag adapted
to bind with a capture probe. In some embodiments, the affinity tag
is selected from the group consisting of His, biotin, and
streptavidin. In some embodiments, each barcode is different. In
some embodiments, the linker comprises a single-stranded nucleic
acid. In some embodiments, the target nucleic acid comprises
genomic DNA.
Methods for Obtaining Haplotype Information
[0184] Some embodiments of the methods and compositions provided
herein include methods of obtaining haplotype information from a
target nucleic acid. Haplotype information can include determining
the presence or absence of different sequences at specified loci in
a target nucleic acid, such as a genome. For example, sequence
information can be obtained for maternal and paternal copies of an
allele. In a polyploidy organism, sequence information can be
obtained for at least one haplotype. Such methods are also useful
in reducing the error rate in obtaining sequence information from
target nucleic acid.
[0185] Generally, methods to obtain haplotype information include
distributing a nucleic acid into one or more compartments such that
each compartment comprises an amount of nucleic acid equivalent to
about a haploid equivalent of the nucleic acid, or equivalent to
less than about a haploid equivalent of the nucleic acid. Sequence
information can then be obtained from each compartment, thereby
obtaining haplotype information. Distributing the template nucleic
acid into a plurality of vessels increases the probability that a
single vessel includes a single copy of an allele or SNP, or that
consensus sequence information obtained from a single vessel
reflects the sequence information of an allele or SNP. As will be
understood, in some such embodiments, a template nucleic acid may
be diluted prior to compartmentalizing the template nucleic acid
into a plurality of vessels. For example, each vessel can contain
an amount of target nucleic acids equal to about a haploid
equivalent of the target nucleic acid. In some embodiments, a
vessel can include less than about one haploid equivalent of a
target nucleic acid.
Method of Haplotyping with Virtual Compartments
[0186] Some methods of obtaining haplotype information provided
herein include the use of virtual compartments. Advantageously,
some such methods enable compartments to include amounts of nucleic
acids equivalent to at least one or more haploid equivalents. In
other words, such methods enable the use of higher concentrations
of nucleic acids in compartments compared to other methods of
haplotyping, thereby increasing the efficiency and yields of
various manipulations.
[0187] In some methods to obtain haplotype information with virtual
compartments, a nucleic acid is compartmentalized into a plurality
of first vessels, and the nucleic acids of each compartment are
provided with a first index; the first-indexed nucleic acids are
combined, and then compartmentalized into a plurality of second
vessels, and the nucleic acids of each compartment are provided
with a second index. A template nucleic acid can be prepared by
undergoing at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of
compartmentalizing, indexing, and pooling. In such a manner, a
template nucleic acid is provided with a plurality of different
indices in a stepwise method. Subsequent to indexing, the indexed
template nucleic acids can be pooled and distributed into a
plurality of compartments such that each compartment is likely to
include an amount of a particular template nucleic acid having a
particular combination of indexes that is equivalent to about a
haploid equivalent of the target nucleic acid, or equivalent to
less than about a haploid equivalent of the target nucleic acid, or
equivalent to more than about a haploid equivalent. In other words,
each vessel can receive an amount of template nucleic acid
comprising more than the equivalent of a haploid equivalent,
however, each copy of an allele or SNP is likely to be associated
with a different combination of indexes. Accordingly, the number of
vessels to compartmentalize a template nucleic acid such that each
vessel includes about an amount of template nucleic acid equivalent
to a haploid or less of a target nucleic acid can be reduced. In
addition, the amount of nucleic acid in each vessel can be greater
than the amount of about a haploid equivalent, thereby increasing
the efficiency and yields of various manipulations.
[0188] There are various methods to index nucleic acids. For
example, in some embodiments, indexes may be inserted into nucleic
acids using transposomes provide herein; indexes can be ligated to
nucleic acids; and indexes can be added to nucleic, acids during
copying, e.g., amplification of a nucleic acid. In some
embodiments, a template nucleic acid comprising an index can be
prepared using transposomes comprising a contiguous transposon
sequence. See e.g., transposome (50) in FIG. 1. Insertion of
contiguous transposon sequences can result in the preservation of
positional information for a particular nucleic acid molecule after
distribution of template nucleic acids between several
compartments. In some embodiments, a template nucleic acid
comprising an index can be prepared using transposomes comprising
non-contiguous transposon sequences. See e.g., transposome (10) in
FIG. 1. Examples of such transposon sequences are set forth in U.S.
Patent Application Publication No. 2010/0120098, which is
incorporated herein by reference in its entirety. Insertion of
non-contiguous transposon sequences can result in the fragmentation
of a particular nucleic acid molecule. Thus, in some embodiments,
insertion of non-contiguous transposon sequences into a template
nucleic acid can reduce positional information for a particular
nucleic acid molecule after distribution of the template nucleic
acids between several compartments. In other words, different
fragments of a particular nucleic acid molecule can be distributed
into different vessels.
[0189] In an example with a diploid genome, after pooling and
dilution in compartments, a greater amount of nucleic acids can be
added to each compartment since the chance of a copy from the
father and copy from the mother of the same region with the same
indexes is lower. For example, one copy of the father and one copy
of the mother for the same region can be present in the same
compartment as long as each contains a different index, for
example, one comes from a transposition reaction with a first index
(index-1) and the other comes from a transposition reaction with a
different first index (index-2). In other words, copies of the same
region/chromosome can be present in the same compartment since
these can be distinguished by their unique index incorporated in
the first transposition reaction. This allows more DNA to be
distributed into each compartment compared to alternative dilution
methods. The dual indexing scheme creates a total number of virtual
compartments of number of initial indexed transposition reactions
multiplied by the number of indexed PCR reactions.
[0190] FIG. 4 depicts an example embodiment of obtaining haplotype
information using virtual compartments. A target nucleic acid
comprising genomic DNA is distributed into a first set of 96
vessels and the nucleic acids of each vessel are provided with a
different first index using a Tn5-derived transposon. Thus a
plurality of first-indexed template nucleic acids is obtained
(e.g., Tn5-1, Tn5-2 . . . , and Tn5-96). The plurality of
first-indexed template nucleic acids are combined and then
redistributed into a second set of 96 vessels and the nucleic acids
of each vessel are provided with a different second index by
amplification of the nucleic acids using primers comprising the
second indexes. Thus a plurality of second-indexed template nucleic
acids is obtained (e.g., PCR1, PCR2 . . . , and PCR96). The
plurality of second-indexed template nucleic acids can be combined
and sequence information obtained. The use of 96.times.96 physical
vessels is equivalent to 9216 virtual compartments.
Methods to Obtain Extended Haplotype Information
[0191] As described above, insertion of non-contiguous transposon
sequences into the template nucleic acid can reduce positional
information for a particular nucleic acid molecule, for example,
after distribution of the template nucleic acids between several
compartments. However, applicant has discovered methods to preserve
such positional information for a particular nucleic acid molecule.
Without being bound to any one theory, it has been observed that
after transposition, the resulting two adjacent fragments of a
particular nucleic acid molecule will tend to be distributed into
the same vessel under conditions that maintain the transposase at
the site of insertion of a transposon sequence. In other words, the
transposase may hold the two resulting two adjacent fragments of a
particular nucleic acid molecule together.
[0192] In some embodiments, a transposase can be removed from a
template nucleic acid subsequent to distributing the template
nucleic in several vessels. A transposase can be removed from the
site of an insertion by various methods well known in the art,
including the addition of a detergent, such as SDS, changing
temperature, Proteinase digestion, chaperone capture and changing
pH. DNA polymerases, with or without strand displacement properties
including, but not limited to, phi29 DNA polymerase, Bst DNA
polymerase, etc. can also be used to dislodge the transposase from
the DNA.
[0193] FIG. 5 depicts an example scheme in which a target nucleic
acid is distributed into a set of first vessels and indexed by
insertion of transposomes, such as transposomes comprising
non-contiguous transposon sequences. The first indexed template
nucleic acids are pooled and distributed into a set of second
vessels and indexed by PCR amplification. Sequence information can
be obtained from the second indexed template nucleic acids.
[0194] Some methods of obtaining extended haplotype information
from a target nucleic acid include (a) obtaining a template nucleic
acid comprising a plurality of transposomes inserted into the
target nucleic acid, wherein at least some of the inserted
transposome each comprise a first transposon sequence, a second
transposon sequence noncontiguous with the first transposon
sequence, and a transposase associated with the first transposon
sequence and the second transposon sequence; (b) compartmentalizing
the template nucleic acid comprising the plurality of inserted
transposomes into each vessel of a plurality of vessels; (c)
removing the transposase from the template nucleic acid; and (d)
obtaining sequence information from the template nucleic acid of
each vessel, thereby obtaining haplotype information from the
target nucleic acid. In some embodiments, compartmentalizing the
template nucleic acid includes providing each vessel with an amount
of template nucleic acid equivalent to greater than about a haploid
equivalent of the target nucleic acid, an amount of template
nucleic acid equivalent to about one haploid equivalent of the
target nucleic acid, or an amount of template nucleic acid
equivalent to less than about a haploid equivalent of the target
nucleic acid.
[0195] An additional embodiment for maintaining contiguity of
target nucleic acids for sequencing applications comprises
utilizing one-sided (i.e., one transposon end) transpositional
events in lieu of two-sided (i.e., two transposon ends)
transpositional events as disclosed herein. For example,
transposases including, but not limited to Mu, MuE392Q mutant, Tn5
have been shown to display one-sided transposition of a transposon
sequence into a target nucleic acid (Haapa et al., 1999, Nucl.
Acids Res. 27(3): 2777-2784). The one-sided transpositional
mechanism of these transposases can be utilized in methods
described here to maintain the contiguity of a sample for
sequencing, for example to haplotype or assemble a target nucleic
acid.
[0196] In one example of one-sided transposition into a target DNA
the transposome, a Tn5 dimer transposase is associated with only
one transposon sequence end. In preferred embodiments, the
transposon end could further comprise additional sequences such as
index sequences, barcodes, and/or primer sequences and the like
which could be used, for example, to identify a sample, amplify or
extend the target nucleic acid and align fragment sequences. The
transposome complex associates with the target nucleic acid, in
that case dsDNA. At the site of transposome association, the
transposase cleaves that strand of the target DNA and inserts the
transposon and any other additional sequences at the point of
cleavage. The transposase remains associated with the target DNA
until it is removes, for example after partitioning of the sample
as described herein the transposase can be removed by degradation
(e.g., use of SDS or other methods as described here). The target
nucleic acid, in this case dsDNA, does not fragment after removal
of the transposase, as such the transposon and any additional
sequences can be incorporated into the target DNA without
fragmenting the DNA. Once the transposase is removed, target
amplification by any means known in the art, in this example single
or multiple primer amplification (due to incorporation of multiple
different primer sequencing included in one or more transposons)
either exponential or linear such as targeted PCR or whole genome
amplification (for example by multiple strand displacement), can be
performed to create libraries for sequencing. As described herein
with respect to the two-sided transposon sequences, a variety of
different combinations of index, barcode, restriction endonuclease
site(s), and/or primer sequence could be included as part of the
transposon sequence depending on the needs of the user. As such,
one-sided transposome complexes could also be utilized to maintain
contiguity of a target nucleic acid for methods disclosed herein
for determining the haplotype of a target nucleic acid.
[0197] One sided transposomes can also be created from the two
transposon/transposase complexes or the looped
transposon/transposase complexes disclosed herein. For example, one
of the transposon sequences of a two transposon complex or one end
of the lopped transposon could be, for example, chemically modified
or blocked so that transposition would not occur, or would
minimally occur, at that end. For example, a dideoxynucleotide, a
hapten such as a biotin could be incorporated at the end of one of
the transposon ends which would inhibit transposition at that end,
thereby allowing for only one transposon, or one end of a looped
transposon, to be inserted into the target nucleic acid.
[0198] In one embodiment, a method of obtaining sequence
information from a target nucleic acid comprises obtaining a
template nucleic acid comprising a plurality of transposons
inserted into said target nucleic acid such that the contiguity of
the template is retained, compartmentalizing the nucleic acids
comprising the plurality of inserted transposons into a plurality
of vessels, generating compartment-specific indexed libraries from
the transposed nucleic acid targets and obtaining sequence
information from the template nucleic acids in each vessel of the
plurality of vessels.
Certain Methods for Preparing Target Nucleic Acids for
Haplotyping
[0199] Some embodiments of the methods and compositions provided
herein include preparing target nucleic acids for haplotyping using
the methods provided herein. Using a pre-amplification method, the
number of unique reads is increased by generating multiple
identical copies of the same nucleic acid fragment as a contiguous
product. In some such embodiments, a library is amplified by
methods such as rolling circle amplification (RCA). In some
embodiments, circular libraries of a target nucleic acid are
prepared and the library amplified by RCA. Such methods generate
extended long nucleic acids.
[0200] An example scheme is shown in FIG. 6. FIG. 6 depicts a
method including preparing target nucleic acids for haplotyping by
generating a library comprising circular molecules by mate-pair and
selection of specific or range of sizes from 1-10 kb or 10-20 kb,
or 20 kb-50 kb, 50-200 kb nucleic acids; amplifying the library by
RCA to generate extended lone nucleic acids; inserting indexes into
the amplified library with transposons; compartmentalizing the
inserted library; removing transposase with SDS; further indexing
the library; and obtaining sequence information from the
library.
[0201] Another example scheme is shown in FIG. 7. FIG. 7 depicts a
method including preparing target nucleic acids for haplotyping by
generating a library comprising circular molecules by hairpin
transposition, gap fill, and selection of a specific size or range
of sizes from 1-10 kb or 10-20 kb, or 20 kb-50 kb, 50-200 kb 5
nucleic acids; amplifying the library by RCA to generate extended
lone nucleic acids; inserting indexes into the amplified library
with transposons; compartmentalizing the inserted library; removing
transposase with SDS; further indexing the library; and obtaining
sequence information from the library.
Methods to Generate Mate-Paired Libraries
[0202] Methods for generating mate-pair libraries include;
fragmenting genomic DNA into large fragments typically greater than
(though not limited to) 1000 bp; circularizing individual fragments
by a method that tags the ligated junction; fragmenting the DNA
further; enriching the tagged junction sequences and ligating
adaptors to the enriched junction sequences so that they may be
sequenced yielding information about the pair of sequences at the
ends of the original long fragment of DNA. These processes involve
at least 2 steps where DNA is fragmented, either physically or
enzymatically. In at least one or more distinct steps, adaptors are
ligated to the ends of fragments. Mate-pair preps typically take
2-3 days to perform and comprise multiple steps of DNA
manipulations. The diversity of the resulting library correlates
directly with the number of steps required to make the library.
[0203] The method provided herein simplifies the number of steps in
the library generation protocol by employing a transposase mediated
reaction that simultaneously fragments and adds adaptor sequences
to the ends of the fragments. At least one or both of the
fragmentation steps (initial fragmentation of genomic DNA and
fragmentation of circularized fragments) may be performed with a
transposome, thus replacing the need for separate fragmentation and
adaptor ligation step. Obviating the polishing, preparation of, and
ligation to fragment ends reduces the number of process steps and
thus increases the yield of usable data in the prep as well as
making the procedure more robust. In one embodiment, the protocol
can be performed without resorting to methods that purify a
selection of sizes based on electrophoresis. This method produces a
broader range of fragment sizes than can be achieved with gel
electrophoretic methods but nonetheless produces usable data. The
advantage is that a labour intensive step is avoided.
[0204] FIG. 8 provides an example scheme where just the initial
fragmentation is replaced with a transposome tagmentation step. The
circularized DNA is fragmented by either physical methods or
chemical/enzymatic methods and the fragments turned into a library
via application of standard sample prep protocols (e.g. TRUSEQ).
FIG. 9 illustrates an example scheme where both the initial
fragmentation and the circularized DNA fragmentation are performed
with a transposome tagmentation step. The adaptor sequences for the
transposome (including the ME sequences) may or may not be
different for the adaptor used for the initial tag mentation and
the subsequent circle tagmentation.
[0205] Amplifying template nucleic acid by generating multiple
copies of each molecule before transposition or introduction of
molecular indexes creates redundancy which can be useful for
getting higher SNP coverage in each haplotype block, and also for
de novo genome assembly, similar to a shotgun approach. Template
nucleic acid can be converted to a defined-size library by
low-frequency transposition, physical shearing, or enzymatic
digestion, and then amplified for a finite number of cycles by
either PCR or a whole genome amplification scheme (for example
using phi29). The amplified library which already contains the
built-in redundancy can be used as the input material for the
haplotyping workflow. This way, every region of the genome is
represented multiple times by multiple copies generated upfront
with each copy contributing a partial coverage of that region;
however, the consensus coverage will be closer to complete.
EXAMPLES
Example 1--Reducing Error Rates
[0206] A library of template nucleic acids was prepared with each
fragment comprising a different barcode. Each fragment was
amplified and sequence information was obtained from at least one
amplified product from each fragment. A consensus sequence was
determined from the sequence information from the amplified
products from each fragment. In particular, a NEXTERA-prepped
sequencing library was sequenced for 500 cycles on a MISEQ
instrument. The library consisted of a distribution of sizes, with
maximum read lengths extending to .about.300 nt. The error rates at
cycle 250 were approximately 15%. If a template was represented
just three times, the error rate dropped to .about.1% at cycle 250.
FIG. 10 illustrates a model of error rates with number of amplified
products sequenced (coverage of each barcode). Error rates decrease
as the number of amplified products sequenced from a fragment
increase.
Example 2--Coupling Transposon Sequences
[0207] This example illustrates methods to couple two transposon
sequences together in various orientations including a 5'-5'
orientation, and a 3'-3' orientation. In an exemplary method,
aldehyde oxyamine is used to form linked oligos via oxime ether
formation. An aldehyde modified oligo (either on the 5' or the 3'
end) is combined with an oligo modified with an oxyamine on the 5'
end in reaction buffer and allowed to incubate for 2 hours. Final
product can be isolated via PAGE purification for example.
[0208] In another exemplary method, bisoxyamine coupling was
performed. Aldehyde modified oligos were dimerized with a
bis-oxyamine (e.g., dioxyamino butane) linker using locally high
concentrations to force bi-substitution. 100-mer oligos were
synthesized with an aldehyde on the 5' end and purified. The
bisoxyamine oligo was synthesized. A 1 mM solution of the
bisoxyamine oligo was made in a low pH reaction buffer containing a
catalyst (5 M urea, 100 mM aniline, 10 mM citrate, 150 mM NaCl, pH
5.6) and added to a 665 .mu.M solution of aldehyde oligo in water.
The entire volume of the solution was diluted 1:1 with reaction
buffer, and allowed to incubate at room temperature for 2 hours. A
titration of various aldehyde:bisoxyamine ratios showed
dimerization at high bisoxyamine ratios. The most successful
conditions were replicated with 3' aldehyde oligos. FIG. 11 shows
results of 5'-5' bisoxyamine coupling reactions in which looped
precursor transposons were observed in the indicated dimer band.
Similar results were observed for 3'-3' bisoxyamine coupling
reactions.
Example 3--Monitoring Transposome Stability
[0209] Transposomes were prepared using long and short transposon
sequences loaded onto transposase. The transposome products
included: A (2 short sequences); B (long and short sequences); and
C (2 long sequences). The relative amounts of each species of
transposome were measured under various conditions, such as
temperature, buffers, ratios of transposon sequences to
transposase. Generally, high NaCl or KCl salt increased exchange of
transposon sequences between transposomes. Glutamate and acetate
buffers eliminated or reduced exchange, with preferred
concentrations between 100-600 mM. Optimum storage conditions were
determined.
Example 4--Maintaining Template Contiguity
[0210] This example illustrates a method for maintaining contiguity
information of a template nucleic acid prepared using transposomes
comprising non-contiguous transposon sequences in which Tn5
transposase stays bound to the template DNA post-transposition.
Target nucleic acid was contacted with transposomes comprising Tn5
transposase, and non-contiguous transposon sequences. FIG. 12 shows
that samples further treated with SDS appeared as a smear of
various fragments of template nucleic acid; samples not treated
with SDS showed retention of putative high molecular weight
template nucleic acid. Thus, even though a nucleic acid may be
fragmented, adjacent sequences may still be associated with one
another by the transposase (as demonstrated by the Tn5 bound DNA
left in the wells).
[0211] In still another exemplary method, a library of template
nucleic acids was prepared using transposomes comprising
non-contiguous transposon sequences with target nucleic acid
comprising human Chromosome 22. FIG. 13 summarizes that haplotype
blocks up to 100 kb were observed for samples in which transposase
was removed by SDS post-dilution. Thus, by practicing methods as
described herein target nucleic acids can maintain target integrity
when transposed, be diluted, and be transformed into sequencing
libraries.
Example 5--Maintaining Template Contiguity
[0212] Target nucleic acids were tagmented with transposomes
comprising non-contiguous transposon sequences (NEXTERA), diluted
to the desired concentration, and then treated with SDS to remove
the transposase enzyme before PCR. As a control, the same amount of
input DNA was tagmented, treated with SDS first and then diluted to
the desired concentration. SDS treatment before dilution removes
proximity information since the transposase enzyme dissociates from
the target DNA with SDS, thereby fragmenting the target DNA. Two
tagmentation reactions were set-up on 50 ng of a Coriell gDNA, 1
reaction was stopped with 0.1% SDS and diluted to 6 pg. Next, the
other reaction was first diluted to 6 pg and then stopped with 0.1%
SDS. The entire reactions were used to set up a 30-cycle PCR
reaction and sequenced on a Gene Analyzer platform (Illumina),
according to the manufacturer's instructions. The reads were mapped
to a human reference genome and the distance distribution was
calculated and plotted.
[0213] As shown in FIG. 14, in the SDS-PostDilution sample, the
median distance shifted to smaller sizes and a large sub-population
of proximally located reads becomes apparent. If there was any
haplotyping, pile up of proximal reads was expected. The bi-modal
distribution of post-dilution sample demonstrated that there is an
enrichment of proximal reads.
[0214] The distance distribution was a measure of sample size (i.e.
the more unique reads, the shorter the distance). The distance
histogram for SDS-PreDilution and SDS-PostDilution samples are
shown in FIG. 14. To correct for the difference in the number of
unique reads, the Pre-Dilution was down-sampled to give the same
number of unique mapped reads (664,741). A significant enrichment
was observed for reads that are immediate neighbors (i.e.
junctions). This was measured by looking at the "distance to next
alignment` distribution and finding the reads which their distance
to their next alignment is the sequence read length minus 9 (which
correspond to 9 bp duplication caused by Tn5 at the insertion
site). Such reads make up 10% of the data (FIG. 15) and with
employment of single primer system for amplification of NEXTERA
libraries, can be doubled. Also implementation of a more
conservative sample prep which allows less sample loss allows
recovery of more junction data. The haplotype resolving power
diminished when input DNA was increased. On the other hand,
reducing the DNA input required more amplification, and therefore
more PCR cycles, generating many PCR duplicates. Using individually
barcoded Tn5 complexes allowed the tagmentation and subsequent
dilutions to be carried out in separate compartments. Low levels of
input from individually barcoded and tagmented material were
combined to elevate the PCR input DNA amounts to the level that
allowed more specific amplification with less waste of sequencing
capacity on redundant reads. Accordingly, using sufficient barcoded
complexes allowed phasing of the majority of the human genome. In
order to increase the haplotype resolving power, barcoding was
implemented at both the complex level and PCR primer level. Such
combinatorial indexing scheme allows the use of very low input DNA
from each individually barcoded complex into PCR reaction, which
would allow powerful haplotype resolution. Using only 40 indexing
oligos (8+12=20 for NEXTERA complexes which generates 8*12=96
individual complexes and 8+12=20 for PCR primers which would allow
8*12=96 additional indexes), 96*96=9216 virtual compartments were
generated for the abovementioned haplotyping workflow. Using a
modified sequencing recipe, all the data was sequenced on a
HiSeq-2000. All 9216 possible barcode combinations were observed in
the sequencing results.
Example 6--Obtaining Haplotype Information with Mu
[0215] Transposomes comprising Mu were used to obtain haplotype
information. 1 ng of genomic DNA was targeted with Mu-TSM in a 50
.mu.l reaction volume with 1.times. TA buffer and 1, 2, 4, or 8
.mu.l of 25 .mu.M Mu-TSM complexes. Reactions were incubated at
37.degree. C. for 2 hours. Samples were diluted to 1 pg/.mu.l. For
Mu inactivation, 10 .mu.l of each sample containing either 1 pg or
5 pg total genomic DNA were prepared. SDS was added to final
concentration of 0.05%. Samples were incubated at 55.degree. C. for
20 minutes. The whole sample was used to set-up a 50 .mu.l PCR
reaction using NPM. PCR was clone for 30 cycles. PCR samples were
cleaned up with 0.6.times. SPRI and resuspended in 20 .mu.l of
re-suspension buffer. Sequencing information was obtained. FIG. 16
shows the observed Pile-up proximal reads observed at sub-haploid
content using transposomes comprising Mu.
[0216] The term "comprising" as used herein is synonymous with
"including," "containing," or "characterized by," and is inclusive
or open-ended and does not exclude additional, unrecited elements
or method steps.
[0217] All numbers expressing quantities of ingredients, reaction
conditions, and so forth used in the specification are to be
understood as being modified in all instances by the term "about."
Accordingly, unless indicated to the contrary, the numerical
parameters set forth herein are approximations that may vary
depending upon the desired properties sought to be obtained. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of any claims in any
application claiming priority to the present application, each
numerical parameter should be construed in light of the number of
significant digits and ordinary rounding approaches.
[0218] The above description discloses several methods and
materials of the present invention. This invention is susceptible
to modifications in the methods and materials, as well as
alterations in the fabrication methods and equipment. Such
modifications will become apparent to those skilled in the art from
a consideration of this disclosure or practice of the invention
disclosed herein. Consequently, it is not intended that this
invention be limited to the specific embodiments disclosed herein,
but that it cover all modifications and alternatives coming within
the true scope and spirit of the invention.
[0219] All references cited herein, including hut not limited to
published and unpublished applications, patents, and literature
references, are incorporated herein by reference in their entirety
and are hereby made a part of this specification. To the extent
publications and patents or patent applications incorporated by
reference contradict the disclosure contained in the specification,
the specification is intended to supersede and/or take precedence
over any such contradictory material.
* * * * *