U.S. patent application number 11/934697 was filed with the patent office on 2009-04-30 for selection of dna adaptor orientation by hybrid capture.
This patent application is currently assigned to Complete Genomics, Inc.. Invention is credited to Arnold Oliphant, Andrew Sparks, George Yeung.
Application Number | 20090111705 11/934697 |
Document ID | / |
Family ID | 39365402 |
Filed Date | 2009-04-30 |
United States Patent
Application |
20090111705 |
Kind Code |
A1 |
Sparks; Andrew ; et
al. |
April 30, 2009 |
SELECTION OF DNA ADAPTOR ORIENTATION BY HYBRID CAPTURE
Abstract
Aspects described and claimed herein provide methods to insert
multiple DNA adaptors into a population of circular target DNAs at
defined positions and orientations with respect to one another by
employing selective capture of defined molecules. The resulting
multi-adaptor constructs are then used in massively-parallel
nucleic acid sequencing techniques.
Inventors: |
Sparks; Andrew; (Los Gatos,
CA) ; Oliphant; Arnold; (Sunnyvale, CA) ;
Yeung; George; (Mountain View, CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS, LLP
ONE MARKET SPEAR STREET TOWER
SAN FRANCISCO
CA
94105
US
|
Assignee: |
Complete Genomics, Inc.
Mountain View
CA
|
Family ID: |
39365402 |
Appl. No.: |
11/934697 |
Filed: |
November 2, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60864992 |
Nov 9, 2006 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/183;
435/320.1; 506/17 |
Current CPC
Class: |
C12N 15/66 20130101;
C12N 15/10 20130101 |
Class at
Publication: |
506/9 ;
435/320.1; 506/17; 435/183 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C12N 15/00 20060101 C12N015/00; C40B 40/08 20060101
C40B040/08; C12N 9/00 20060101 C12N009/00 |
Claims
1. A method for selecting for orientation of two adaptors with
respect to one another in library constructs comprising: (a)
obtaining target nucleic acid; (b) ligating a first adaptor to the
target nucleic acid to produce first library constructs, wherein
one strand of the first adaptor comprises a capture sequence; (c)
ligating first and second arms of a second adaptor to the
linearized first library constructs to form second library
constructs, wherein at least one strand of one of the second
adaptor arms comprises a functional group; (d) capturing
functionalized double-stranded second library constructs and
discarding un-functionalized second library constructs; (e) eluting
single-stranded nucleic acids without a functional group from the
captured double-stranded functionalized second library constructs;
and (f) capturing the capture sequence in the one strand of the
first adaptor, thereby selecting for library constructs having a
desired orientation of the second adaptor with respect to the first
adaptor.
2. The method of claim 1, further comprising repeating processes
(b) through (f) until a desired number of adaptors have been
inserted into the nucleic acid library constructs.
3. The method of claim 1, wherein the first library constructs are
cut with a restriction endonuclease after being circularized.
4. The method of claim 1, wherein the first adaptor is ligated to
the target nucleic acid as two adaptor arms.
5. The method of claim 1, wherein the functional group is biotin
and the functionalized double-stranded second library constructs
are captured by a strepavidin column.
6. The method of claim 1, wherein the first and second adaptors
further comprise Type IIs endonuclease recognition sites.
7. A method for selecting for orientation of two adaptors with
respect to one another in library constructs comprising: (a)
obtaining a target nucleic acid; (b) ligating a first adaptor to
the target nucleic acid to produce first library constructs,
wherein one strand of the first adaptor comprises a capture
sequence; (c) ligating first and second arms of a second adaptor to
the linearized first library constructs to form second library
constructs; (d) amplifying the second library constructs with a
functionalized primer complementary to an end of one strand of the
second adaptor; (d) capturing functionalized amplified
double-stranded second library constructs and discarding
unfunctionalized second library constructs; (e) eluting
single-stranded nucleic acids without a functional group from the
captured double-stranded functionalized second library constructs;
and (f) capturing the capture sequence in the one strand of the
first adaptor, thereby selecting for library constructs having a
desired orientation of the second adaptor with respect to the first
adaptor.
8. The method of claim 7, wherein the first library constructs are
cut with a restriction endonuclease after being circularized.
9. The method of claim 7, wherein each adaptor is ligated to the
target nucleic acid as two adaptor arms.
10. The method of claim 7, wherein the functional group is biotin
and the functionalized double-stranded second library constructs
are captured by a strepavidin column.
11. The method of claim 7, wherein each adaptor further comprises
one or more endonuclease recognition sites.
12. The method of claim 11, wherein the endonuclease recognition
sites are Type IIs endonuclease recognition sites.
13. An amplicon made by amplification of a circular library
construct comprising target nucleic acid interspersed with a
plurality of adaptors, wherein at least one of the plurality of
adaptors has a desired orientation with respect to at least one of
the other of the plurality of adaptors.
14. The amplicon of claim 13, wherein each of the plurality of
adaptors has a desired orientation with respect to at least one
other of the plurality of adaptors.
15. The amplicon of claim 13, wherein one or more of the adaptors
comprises a restriction endonuclease recognition site.
16. The amplicon of claim 15, wherein the restriction endonuclease
recognition site is a Type IIs restriction endonuclease recognition
site.
17. The amplicon of claim 13, wherein each adaptor of the plurality
of adaptors further comprise a different anchor primer binding site
at a 5' and 3' end of each of the plurality of adaptors.
18. A multiplicity of amplicons of circular library constructs,
wherein each amplicon comprises target nucleic acid interspersed
with a plurality of adaptors, wherein at least one of the plurality
of adaptors has a desired orientation with respect to at least one
of the other of the plurality of adaptors.
19. The multiplicity of amplicons of claim 18, wherein each of the
plurality of adaptors has a desired orientation with respect to at
least one other of the plurality of adaptors.
20. The multiplicity of amplicons of claim 18, wherein the target
nucleic acid is genomic DNA, cDNA or RNA, and wherein the
multiplicity of amplicons comprises substantially all of genomic
DNA, cDNA or RNA of interest.
21. The multiplicity of amplicons of claim 18, wherein one or more
of the adaptors comprises a restriction endonuclease recognition
site.
22. The multiplicity of amplicons of claim 21, wherein the
restriction endonuclease recognition site is a Type IIs restriction
endonuclease recognition site.
23. The multiplicity of amplicons of claim 18, wherein each adaptor
of the plurality of adaptors further comprise a different anchor
primer binding site at a 5' and 3' end of each of the plurality of
adaptors.
24. A kit for inserting interspersed adaptors in target nucleic
acid, wherein said kit comprises: b) a first double-stranded
adaptor; c) a functionalized second double stranded adaptor; d)
reagents for capturing functionalized second double-stranded
adaptor; and (e) reagents for capturing one strand of the first
double-stranded adaptor.
25. The kit of claim 24 further comprising: a) a ligase; b) a first
Type IIs restriction endonuclease; c) a second Type IIs restriction
endonuclease; or d) a functionalized third adaptor.
Description
[0001] This application claims priority to U.S. Provisional
Application 60/864,992 filed Nov. 9, 2006.
BACKGROUND
[0002] Large-scale sequence analysis of genomic DNA is central to
understanding a wide range of biological phenomena related to
health and disease in humans and is economically important plants
and animals. The need for low-cost, high-throughput sequencing and
re-sequencing has led to the development of new approaches to
sequencing that employ parallel analysis of many target DNA
fragments simultaneously. Improvements to sequencing methods and
increasing the amount and quality of data from such methods is of
great value in the art.
SUMMARY
[0003] Embodiments described and claimed herein address the
foregoing and other situations by providing methods to provide
repeated cycles of nucleic acid cleavage and ligation to insert
multiple DNA adaptors into a population of circular target DNAs at
defined positions and orientations with respect to one another. The
resulting multi-adaptor constructs are then used in
massively-parallel nucleic acid sequencing techniques.
[0004] Aspects of the technology provide methods for selecting for
orientation of two adaptors with respect to one another in library
constructs comprising: obtaining target nucleic acid; ligating a
first adaptor to the target nucleic acid to produce first library
constructs wherein one strand of the first adaptor comprises a
capture sequence; ligating first and second arms of a second
adaptor to the linearized first library constructs to form second
library constructs, wherein wherein at least one strand of the
second adaptor arm comprises a functional group; capturing
functionalized double-stranded second library constructs and
discarding un-functionalized second library constructs; denaturing
and eluting single-stranded nucleic acids from the captured
double-stranded functionalized second library constructs; and
capturing the capture sequence in the one strand of the first
adaptor, thereby selecting for orientation of the second adaptor
with respect to the first adaptor in the library constructs.
[0005] Other aspects of the technology provide methods for
selecting for orientation of two adaptors with respect to one
another in library constructs comprising: obtaining target nucleic
acid; ligating a first adaptor to the target nucleic acid to
produce first library constructs; ligating first and second arms of
a second adaptor to the linearized first library constructs to form
second library constructs; amplifying the second library construct
with a functionalized primer complementary to an end of one strand
of the second adaptor; capturing functionalized amplified
double-stranded second library constructs and discarding un-
functionalized second library constructs; denaturing and eluting
single-stranded nucleic acids from the captured double-stranded
functionalized second library constructs; and capturing the capture
sequence in the one strand of the first adaptor, thereby selecting
for orientation of the second adaptor with respect to the first
adaptor in the library constructs.
[0006] In some aspects of this method, the first library constructs
are circularized between the two ligating steps. In other aspects,
the first library constructs are cut with a restriction
endonuclease after being circularized. In yet other aspects, the
first adaptor is ligated to the target nucleic acid as two adaptor
arms. Also, in other aspects the first and second adaptors further
comprise Type IIs endonuclease recognition sites.
[0007] In other aspects, two-component binding systems with high
affinity and specificity (e.g., avidin-biotin and antibody-hapten
systems) can be used in the immobilization and isolation of certain
constructs. One functional group of the pair can be attached to an
adaptor for use in the isolation of molecules comprising that
adaptor. For example, the functional group can be biotin and the
functionalized double-stranded second library constructs can be
captured by strepavidin.
[0008] Another aspect of the invention provides a method for
selecting for orientation of two or more adaptors with respect to
one another in library constructs comprising: (a) obtaining target
nucleic acid; (b) ligating a first adaptor to the target nucleic
acid to produce first library constructs wherein one strand of the
first adaptor comprises a capture sequence; (c) ligating first and
second arms of a second adaptor to the linearized first library
construct to form second library constructs, wherein at least one
end of the second adaptor arm comprises; (d) capturing
functionalized double-stranded second library constructs and
discarding un-functionalized second library constructs; (e)
denaturing and eluting single-stranded nucleic acids from the
captured double-stranded functionalized second library constructs;
(f) capturing the capture sequence in the one strand of the first
adaptor, thereby selecting for orientation of the second adaptor to
the first adaptor in the library constructs; (g) repeating
processes (b) through (f) until a desired number of adaptors have
been inserted into the nucleic acid library constructs.
[0009] In some aspects, either the first or second adaptor arms of
the second adaptor that is ligated to the first library construct
comprises a functional group; in other aspects, after ligation of
the first and second arms of the second adaptor, the second library
constructs are amplified with a functionalized primer complementary
to an end of one strand of the second adaptor, and the
functionalized amplified double-stranded second library constructs
are captured.
[0010] Also in some aspects, amplicons made by sequentially
selectively capturing a functionalized adaptor and selectively
capturing one strand of one adaptor in a library construct are
provided, as are libraries comprising a multiplicity (five or more)
of such amplicons. In other aspects, kits are provided for
selecting for desired orientations of multiple adaptors in library
constructs comprising a first double-stranded adaptor; a
functionalized second double stranded adaptor; reagents for
capturing the functionalized second double stranded adaptor; and
reagents for capturing one strand of the first double-stranded
adaptor.
[0011] This Summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This Summary is not intended to identify
key or essential features of the claimed subject matter, nor is it
intended to be used to limit the scope of the claimed subject
matter. Other features, details, utilities, and advantages of the
claimed subject matter will be apparent from the following written
Detailed Description including those aspects illustrated in the
accompanying drawings and defined in the appended claims.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0012] FIG. 1 is a simplified flow diagram of an overall method for
sequencing nucleic acids using the processes of the claimed
invention.
[0013] FIG. 2 is a schematic representation of one aspect of a
method for assembling adaptor/target nucleic acid library
constructs.
[0014] FIG. 3 is a schematic illustration of a basic adaptor
insertion process.
[0015] FIG. 4 is a schematic illustration of one aspect of a DNA
array employing multi-adaptor nucleic acid library constructs.
[0016] FIG. 5 is a schematic illustration of the components that
may be used in an exemplary sequencing-by-ligation technique.
[0017] FIG. 6 is a schematic illustration of an insertion of a
second adaptor relative to a first adaptor in a nucleic acid
library construct.
[0018] FIG. 7 is a schematic representation of components of an
exemplary adaptor useful for selecting insertion orientation.
[0019] FIG. 8 is a schematic representation of adaptor insertion
allowing subsequent circularization of the target/adaptor
construct.
[0020] FIG. 9 is a schematic illustration of a hybrid capture
process for selecting constructs where adaptors are inserted into a
target nucleic acid in a desired orientation.
DEFINITIONS
[0021] The practice of the techniques described herein may employ,
unless otherwise indicated, conventional techniques and
descriptions of organic chemistry, polymer technology, molecular
biology (including recombinant techniques), cell biology,
biochemistry, and sequencing technology, which are within the skill
of those who practice in the art. Such conventional techniques
include polymer array synthesis, hybridization and ligation of
polynucleotides, and detection of hybridization using a label.
Specific illustrations of suitable techniques can be had by
reference to the examples herein. However, other equivalent
conventional procedures can, of course, also be used. Such
conventional techniques and descriptions can be found in standard
laboratory manuals such as Green, et al., Eds. (1999), Genome
Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel,
Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual;
Dieffenbach, Dveksler, Eds. (2003), PCR Primer: A Laboratory
Manual; Bowtell and Sambrook (2003), DNA Microarrays: A Molecular
Cloning Manual; Mount (2004), Bioinformatics: Sequence and Genome
Analysis; Sambrook and Russell (2006), Condensed Protocols from
Molecular Cloning: A Laboratory Manual; and Sambrook and Russell
(2002), Molecular Cloning: A Laboratory Manual (all from Cold
Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry
(4th Ed.) W.H. Freeman, New York N.Y.; Gait, "Oligonucleotide
Synthesis: A Practical Approach" 1984, IRL Press, London; Nelson
and Cox (2000), Lehninger, Principles of Biochemistry 3.sup.rd Ed.,
W.H. Freeman Pub., New York, N.Y.; and Berg et al. (2002)
Biochemistry, 5.sup.th Ed., W.H. Freeman Pub., New York, N.Y., all
of which are herein incorporated in their entirety by reference for
all purposes.
[0022] Note that as used herein and in the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "an agent" refers to one agent or mixtures of agents,
and reference to "the method of administration" includes reference
to equivalent steps and methods known to those skilled in the art,
and so forth.
[0023] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications mentioned herein are incorporated herein by reference
for the purpose of describing and disclosing devices, formulations
and methodologies which are described in the publication and which
might be used in connection with the presently described
invention.
[0024] Where a range of values is provided, it is understood that
each intervening value, between the upper and lower limit of that
range and any other stated or intervening value in that stated
range is encompassed within the invention. The upper and lower
limits of these smaller ranges may independently be included in the
smaller ranges, and are also encompassed within the invention,
subject to any specifically excluded limit in the stated range.
Where the stated range includes one or both of the limits, ranges
excluding either both of those included limits are also included in
the invention.
[0025] In the following description, numerous specific details are
set forth to provide a more thorough understanding of the present
invention. However, it will be apparent to one of skill in the art
that the present invention may be practiced without one or more of
these specific details. In other instances, well-known features and
procedures well known to those skilled in the art have not been
described in order to avoid obscuring the invention.
[0026] "Adaptor" refers to an engineered construct comprising
"adaptor elements" where one or more adaptors may be interspersed
within target nucleic acid in a library construct. The adaptor
elements or features included in any adaptor vary widely depending
on the use of the adaptors, but typically include sites for
restriction endonuclease recognition and/or cutting, sites for
primer binding (for amplifying the library constructs) or anchor
primer binding (for sequencing the target nucleic acids in the
library constructs), nickase sites, and the like. In some aspects,
adaptors are engineered so as to comprise one or more of the
following: 1) a length of about 20 to about 250 nucleotides, or
about 40 to about 100 oligonucleotides, or less than about 60
nucleotides, or less than about 50 nucleotides; 2) features so as
to be ligated to the target nucleic acid as two "arms"; 3)
different and distinct anchor binding sites at the 5' and the 3'
ends of the adaptor for use in sequencing of adjacent target
nucleic acid; and 4) one or more restriction sites.
[0027] "Amplicon" means the product of a polynucleotide
amplification reaction. That is, it is a population of
polynucleotides that are replicated from one or more starting
sequences. Amplicons may be produced by a variety of amplification
reactions, including but not limited to polymerase chain reactions
(PCRs), linear polymerase reactions, nucleic acid sequence-based
amplification, circle dependant amplification and like reactions
(see, e.g., U.S. Pat. Nos. 4,683,195; 4,965,188; 4,683,202;
4,800159; 5,210,015; 6,174,670; 5,399,491; 6,287,824 and 5,854,033;
and US Pub. No. 2006/0024711).
[0028] "Circle dependant replication" or "CDR" refers to multiple
displacement amplification of a double-stranded circular template
using one or more primers annealing to the same strand of the
circular template to generate products representing only one strand
of the template. In CDR, no additional primer binding sites are
generated and the amount of product increases only linearly with
time. The primer(s) used may be of a random sequence (e.g., one or
more random hexamers) or may have a specific sequence to select for
amplification of a desired product. Without further modification of
the end product, CDR often results in the creation of a linear
construct having multiple copies of a strand of the circular
template in tandem, i.e. a linear, single-stranded concatamer of
multiple copies of a strand of the template.
[0029] "Circle dependant amplification" or "CDA" refers to multiple
displacement amplification of a double-stranded circular template
using primers annealing to both strands of the circular template to
generate products representing both strands of the template,
resulting in a cascade of multiple-hybridization, primer-extension
and strand-displacement events. This leads to an exponential
increase in the number of primer binding sites, with a consequent
exponential increase in the amount of product generated over time.
The primers used may be of a random sequence (e.g., random
hexamers) or may have a specific sequence to select for
amplification of a desired product. CDA results in a set of
concatemeric double-stranded fragments is formed.
[0030] "Complementary" or "substantially complementary" refers to
the hybridization or base pairing or the formation of a duplex
between nucleotides or nucleic acids, such as, for instance,
between the two strands of a double-stranded DNA molecule or
between an oligonucleotide primer and a primer binding site on a
single-stranded nucleic acid. Complementary nucleotides are,
generally, A and T (or A and U), or C and G. Two single-stranded
RNA or DNA molecules are said to be substantially complementary
when the nucleotides of one strand, optimally aligned and compared
and with appropriate nucleotide insertions or deletions, pair with
at least about 80% of the other strand, usually at least about 90%
to about 95%, and even about 98% to about 100%.
[0031] "Duplex" means at least two oligonucleotides or
polynucleotides that are fully or partially complementary and which
undergo Watson-Crick type base pairing among all or most of their
nucleotides so that a stable complex is formed. The terms
"annealing" and "hybridization" are used interchangeably to mean
formation of a stable duplex. "Perfectly matched" in reference to a
duplex means that the poly- or oligonucleotide strands making up
the duplex form a double-stranded structure with one another such
that every nucleotide in each strand undergoes Watson-Crick base
pairing with a nucleotide in the other strand. A "mismatch" in a
duplex between two oligonucleotides or polynucleotides means that a
pair of nucleotides in the duplex fails to undergo Watson-Crick
basepairing.
[0032] "Hybridization" refers to the process in which two
single-stranded polynucleotides bind non-covalently to form a
stable double-stranded polynucleotide. The resulting (usually)
double-stranded polynucleotide is a "hybrid" or "duplex."
"Hybridization conditions" will typically include salt
concentrations of less than about 1M, more usually less than about
500 mM and may be less than about 200 mM. A "hybridization buffer"
is a buffered salt solution such as 5% SSPE, or other such buffers
known in the art. Hybridization temperatures can be as low as
5.degree. C., but are typically greater than 22.degree. C., and
more typically greater than about 30.degree. C., and typically in
excess of 37.degree. C. Hybridizations are usually performed under
stringent conditions, i.e., conditions under which a probe will
hybridize to its target subsequence but will not hybridize to the
other, uncomplimentary sequences. Stringent conditions are
sequence-dependent and are different in different circumstances.
For example, longer fragments may require higher hybridization
temperatures for specific hybridization than short fragments. As
other factors may affect the stringency of hybridization, including
base composition and length of the complementary strands, presence
of organic solvents, and the extent of base mismatching, the
combination of parameters is more important than the absolute
measure of any one parameter alone. Generally stringent conditions
are selected to be about 5.degree. C. lower than the T.sub.m for
the specific sequence at a defined ionic strength and pH. Exemplary
stringent conditions include a salt concentration of at least 0.01M
to no more than 1M sodium ion concentration (or other salt) at a pH
of about 7.0 to about 8.3 and a temperature of at least 25.degree.
C. For example, conditions of 5.times.SSPE (750 mM NaCl, 50 mM
sodium phosphate, 5 mM EDTA at pH 7.4) and a temperature of
30.degree. C. are suitable for allele-specific probe
hybridizations.
[0033] "Ligation" means to form a covalent bond or linkage between
the termini of two or more nucleic acids, e.g., oligonucleotides
and/or polynucleotides, in a template-driven reaction. The nature
of the bond or linkage may vary widely and the ligation may be
carried out enzymatically or chemically. As used herein, ligations
are usually carried out enzymatically to form a phosphodiester
linkage between a 5' carbon terminal nucleotide of one
oligonucleotide with a 3' carbon of another nucleotide. Template
driven ligation reactions are described in the following
references: U.S. Pat. Nos. 4,883,750; 5,476,930; 5,593,826; and
5,871,921.
[0034] "Microarray" or "array" refers to a solid phase support
having a surface, preferably but not exclusively a planar or
substantially planar surface, which carries an array of sites
containing nucleic acids such that each site of the array comprises
identical copies of oligonucleotides or polynucleotides and is
spatially defined and not overlapping with other member sites of
the array; that is, the sites are spatially discrete. The array or
microarray can also comprise a non-planar interrogatable structure
with a surface such as a bead or a well. The oligonucleotides or
polynucleotides of the array may be covalently bound to the solid
support, or may be non-covalently bound. Conventional microarray
technology is reviewed in, e.g., Schena, Ed. (2000), Microarrays: A
Practical Approach (IRL Press, Oxford). As used herein, "random
array" or "random microarray" refers to a microarray where the
identity of the oligonucleotides or polynucleotides is not
discernable, at least initially, from their location but may be
determined by a particular operation on the array, such as by
sequencing, hybridizing decoding probes or the like. See, e.g.,
U.S. Pat. Nos. 6,396,995; 6,544,732; 6,401,267; and 7,070,927; WO
publications WO 2006/073504 and 2005/082098; and US Pub Nos.
2007/0207482 and 2007/0087362.
[0035] "Nucleic acid", "oligonucleotide", "polynucleotide", "oligo"
or grammatical equivalents used herein refers generally to at least
two nucleotides covalently linked together. A nucleic acid
generally will contain phosphodiester bonds, although in some cases
nucleic acid analogs may be included that have alternative
backbones such as phosphoramidite, phosphorodithioate, or
methylphophoroamidite linkages; or peptide nucleic acid backbones
and linkages. Other analog nucleic acids include those with
bicyclic structures including locked nucleic acids, positive
backbones, non-ionic backbones and non-ribose backbones.
Modifications of the ribose-phosphate backbone may be done to
increase the stability of the molecules; for example, PNA:DNA
hybrids can exhibit higher stability in some environments.
[0036] "Primer" means an oligonucleotide, either natural or
synthetic, that is capable, upon forming a duplex with a
polynucleotide template, of acting as a point of initiation of
nucleic acid synthesis and being extended from its 3' end along the
template so that an extended duplex is formed. The sequence of
nucleotides added during the extension process is determined by the
sequence of the template polynucleotide. Primers usually are
extended by a DNA polymerase.
[0037] "Probe" means generally an oligonucleotide that is
complementary to an oligonucleotide or target nucleic acid under
investigation. Probes used in certain aspects of the claimed
invention are labeled in a way that permits detection, e.g., with a
fluorescent or other optically-discernable tag.
[0038] "Sequence determination" in reference to a target nucleic
acid means determination of information relating to the sequence of
nucleotides in the target nucleic acid. Such information may
include the identification or determination of partial as well as
full sequence information of the target nucleic acid. The sequence
information may be determined with varying degrees of statistical
reliability or confidence. In one aspect, the term includes the
determination of the identity and ordering of a plurality of
contiguous nucleotides in a target nucleic acid starting from
different nucleotides in the target nucleic acid.
[0039] "Target nucleic acid" means a nucleic acid from a gene, a
regulatory element, genomic DNA, cDNA, RNAs including mRNAs, rRNAs,
siRNAs, miRNAs and the like and fragments thereof. A target nucleic
acid may be a nucleic acid from a sample, or a secondary nucleic
acid such as a product of an amplification reaction.
[0040] As used herein, the term "T.sub.m" is commonly defined as
the temperature at which half of the population of double-stranded
nucleic acid molecules becomes dissociated into single strands. The
equation for calculating the Tm of nucleic acids is well known in
the art. As indicated by standard references, a simple estimate of
the Tm value may be calculated by the equation:
T.sub.m=81.5+16.6(log10[Na+])0.41(%[G+C])-675/n-1.0m, when a
nucleic acid is in aqueous solution having cation concentrations of
0.5 M, or less, the (G+C) content is between 30% and 70%, n is the
number of bases, and m is the percentage of base pair mismatches
(see e.g., Sambrook J et al., "Molecular Cloning, A Laboratory
Manual", 3rd Edition, Cold Spring Harbor Laboratory Press (2001)).
Other references include more sophisticated computations, which
take structural as well as sequence characteristics into account
for the calculation of T.sub.m (see also, Anderson and Young
(1985), Quantitative Filter Hybridization, Nucleic Acid
Hybridization, and Allawi and SantaLucia (1997), Biochemistry
36:10581-94).
DETAILED DESCRIPTION
[0041] Technology is described herein for providing nucleic acid
constructs having interspersed adaptors inserted in a desired
orientation with respect to one another for use in large scale
sequencing methods. Many adaptor insertion methods developed to
date do not allow control of the orientation of newly inserted
adaptors vis-a-vis previously inserted adaptors. The inability to
control the orientation of adaptors with respect to one another can
have a number of undesired consequences. The presence of adaptors
in both orientations in a population of target nucleic acid/adaptor
library constructs may require multiple sequencing primers in each
sequencing reaction to enable sequencing regardless of the
orientation of a given adaptor. In addition, analysis of sequence
data collected from multiple adaptors of unspecified orientation
may require either determination of the orientation of each adaptor
or consideration of all possible combinations of adaptor
orientation during assembly.
Overview of Sequencing Approaches for use with the Claimed
Invention
[0042] FIG. 1 is a simplified flow diagram of an overall method 100
for sequencing nucleic acids using the processes of the claimed
invention. Generally, creation of a target molecule for sequencing
is accomplished by extracting and preparing target nucleic acids
110 (e.g., fractionating, shearing or cleaving), constructing a
library with the sheared target nucleic acids using engineered
adaptors 120, replicating the library constructs to form amplified
library constructs (e.g., forming DNA nanoballs through circle
dependant replication) 130, and sequencing the amplified target
nucleic acids.
[0043] In process 110 of method 100, the target nucleic acids for
some aspects are derived from genomic DNA. In some aspects such as
whole genome sequencing, 10-100 genome-equivalents of DNA
preferably are obtained to ensure that the population of target DNA
fragments covers the entire genome. The target genomic DNA is
isolated using conventional techniques, for example as disclosed in
Sambrook and Russell, Molecular Cloning: A Laboratory Manual. The
target genomic DNA is then fragmented to a desired size by
conventional techniques including enzymatic digestion, shearing, or
sonication. Fragment size of the target nucleic acid can vary
depending on the source target nucleic acid and the library
construction methods used, but typically range from 50 nucleotides
in length to over 11 kb in length, including 200-700 nucleotides in
length, 400-600 nucleotides in length, 450-550 in length, or 4 kb
to over 10 kb in length. Alternatively, in some aspects, the target
nucleic acids comprise mRNAs or cDNAs. In specific embodiments, the
target DNA is created using isolated transcripts from a biological
sample. Isolated mRNA may be reverse transcribed into cDNAs using
conventional techniques, again as described in Genome Analysis: A
Laboratory Manual Series (Vols. I-IV) or Molecular Cloning: A
Laboratory Manual.
[0044] In process 120 of method 100, a library is constructed using
the fragmented target nucleic acids. Library construction will be
discussed in detail infra; briefly, the library constructs are
assembled by inserting adaptor molecules at a multiplicity of sites
throughout each target nucleic acid fragment. The interspersed
adaptors permit acquisition of sequence information from multiple
sites in the target nucleic acid consecutively or simultaneously.
In some aspects, the interspersed adaptors are inserted at
intervals within a contiguous region of the target nucleic acids at
predetermined positions. The intervals may or may not be equal. In
some aspects, the accuracy of the spacing between interspersed
adaptors may be known only to an accuracy of one to a few
nucleotides. In other aspects, the spacing of the adaptors is
known, and the orientation of each adaptor relative to other
adaptors in the library constructs is known.
[0045] In process 130 of method 100, the library constructs are
amplified and, in some aspects, are replicated to form DNA
nanoballs. In such a process, the library constructs (the target
nucleic acids with the interspersed adaptors) are replicated in
such a way so as to form single-stranded DNA concatemers of each
library construct, each concatamer comprising multiple linear
tandem repeats of the library construct. Single-stranded DNA
concatemers under conventional conditions (in buffers, e.g., TE,
SSC, SSPE or the like) form random coils in a manner known in the
art (e.g., see Edvinssom (2002), "On the size and shape of polymers
and polymer complexes," Dissertation 696 (University of Uppsala)).
Concatemeric DNA randomly coiled forms nanoballs (also termed "DNA
nanoballs", "nucleic acid nanoballs" or "DNBs").
[0046] In process 140 of method 100, the DNBs formed in process 130
are sequenced. In some aspects, the DNBs are randomly arrayed on a
planar surface. The DNBs may be covalently or noncovalently
attached to the planar surface. The target nucleic acids within
each DNB are then sequenced by iterative interrogation using
sequencing-by-synthesis techniques and/or sequencing-by-ligation
techniques.
[0047] FIG. 2 is a schematic representation of one aspect of a
method for assembling adaptor/target nucleic acid library
constructs. DNA, such as genomic DNA 202, is isolated and
fragmented 203 into target nucleic acids 204 using standard
techniques as described briefly above. The fragmented target
nucleic acids 204 are then repaired so that the 5' and 3' ends of
each strand are flush or blunt ended. Following this reaction, each
fragment is "A-tailed" with a single A added to the 3' end of each
strand of the fragmented target nucleic acids using a
non-proofreading polymerase 205. Also as part of process 205, a
first and second arm of a first adaptor is then ligated to each
target nucleic acid, producing a target nucleic acid with adaptor
arms ligated to each end 206. In one aspect, the adaptor arms are
"T tailed" to be complementary to the A tailing of the target
nucleic acid, facilitating ligation of the adaptor arms in a known
orientation.
[0048] In a preferred embodiment, the invention provide adaptor
ligation to each fragment in a manner that minimizes the creation
of intra- or intermolecular ligation artefacts. This is desirable
because random fragments of target nucleic acids forming ligation
artefacts with one another create false proximal genomic
relationships between target nucleic acid fragments, complicating
the sequence alignment process. The aspect shown in FIG. 2 shows
step 205 as a combination of blunt end repair and an A tail
addition. This preferred aspect using both A tailing and T tailing
to attach the adaptor to the DNA fragments prevents random intra-
or inter-molecular associations of adaptors and fragments, which
reduces artefacts that would be created from self-ligation,
adaptor-adaptor or fragment-fragment ligation.
[0049] As an alternative to A tailing, various other methods can be
implemented to prevent formation of ligation artefacts of the
target nucleic acids and the adaptors, as well as orient the
adaptor arms with respect to the target nucleic acids, including
using complementary NN overhangs in the target nucleic acids and
the adaptor arms, or employing blunt end ligation with an
appropriate target nucleic acid to adaptor ratio to optimize single
fragment nucleic acid/adaptor arm ligation ratios.
[0050] In process 207, the linear target nucleic acid 206 is
circularized, a process that will be discussed in detail infra,
resulting in a circular library construct 208 comprising target
nucleic acid and an adaptor. Note that the circularization process
results in bringing the first and second arms of the first adaptor
together to form a contiguous adaptor sequence in the circular
construct. In process 209, the circular construct is amplified,
such as by circle dependant amplification, using, e.g., random
hexamers and .phi.29 or helicase. Alternatively, target nucleic
acid/adaptor structure 206 may remain linear, and amplification may
be accomplished by PCR primed from sites in the adaptor arms. The
amplification 209 preferably is a controlled amplification process
and uses a high fidelity, proof-reading polymerase, resulting in a
sequence-accurate library of amplified target nucleic acid/adaptor
constructs where there is sufficient representation of the genome
or one or more portions of the genome being queried.
[0051] In aspects herein, the first adaptor comprises two Type IIs
restriction endonuclease recognition sites, positioned such that
the target nucleic acid outside the recognition sequence (and
outside of the adaptor) is cut 210. The arrows around structure 210
indicate the recognition sites and the site of restriction. In
process 211, EcoP15, a Type IIs restriction endonuclease, is used
to cut the library constructs. Note that in the aspect shown in
FIG. 2, a portion of each library construct mapping to a portion of
the target nucleic acid will be cut away from the construct (the
portion of the target nucleic acid between the arrow heads in
structure 210). Restriction of the library constructs with EcoP15
in process 211 results in a library of linear constructs containing
the first adaptor, with the first adaptor "interior" to the ends of
the linear construct 212. The resulting linear library construct
will have a size defined by the distance between the endonuclease
recognition sites and the endonuclease restriction site plus the
size of the adaptor. In process 213, the linear construct 212, like
the fragmented target nucleic acid 204, is treated by conventional
methods to become blunt or flush ended, A tails comprising a single
A are added to the 3' ends of the linear library construct using a
non-proofreading polymerase and first and second arms of a second
adaptor are ligated to ends of the linearized library construct by
A-T tailing and ligation 213. The resulting library construct
comprises the structure seen at 214, with the first adaptor
interior to the ends of the linear construct, with target nucleic
acid flanked on one end by the first adaptor, and on the other end
by either the first or second arm of the second adaptor.
[0052] In process 215, the double-stranded linear library
constructs are treated so as to become single-stranded 216, and the
single-stranded library constructs 216 are then ligated 217 to form
single-stranded circles of target nucleic acid interspersed with
two adaptors 218. The ligation/circularization process of 217 is
performed under conditions that optimize intramolecular
ligation.
[0053] Next, in the two-adaptor aspect shown in FIG. 2, the
single-stranded, circularized library constructs 218 are amplified
by circle dependant replication 219 to form DNA nanoballs 220.
Circle dependant replication is performed, e.g., using specific
primers where the amplification product displaces its own tail,
producing linear, tandem single-stranded copies of |-target nucleic
acid/adaptor 1/target nucleic acid/adaptor 2-| library constructs.
As the tandem copies begin to multiply, the library constructs
begin to coil and form secondary structures, ultimately forming DNA
nanoballs. Each library construct contains in some aspects between
about ten to about 5000 copies, or from about 250 copies to about
2500 copies of the |-target nucleic acid/adaptor 1/target nucleic
acid/adaptor 2-| repeats, and preferably contains about 500 to
about 1200 copies of the the |-target nucleic acid/adaptor 1/target
nucleic acid/adaptor 2-| repeats The resulting DNA nanoballs 220,
then, are clonal populations of DNA in discrete structures, which
can then be arrayed and sequenced (process not shown).
[0054] FIG. 3 is a simplified schematic illustration showing the
cyclical nature of the basic adaptor insertion process 300 where
two, three, four, five or more adaptors can be inserted into a
target nucleic acid. A fragmented target nucleic acid is shown at
302. Process 303 provides adaptor arm to target nucleic acid
ligation (as was described with some detail in the discussion of
the aspect shown in FIG. 2), resulting in a linear target nucleic
acid with first and second adaptor arms of a first adaptor ligated
onto its ends 304. The adaptor arms are then ligated to one another
in an intramolecular reaction that results in a circularization of
the target nucleic acid/adaptor library construct 306. The library
construct is then amplified 307 resulting in a population
comprising a plurality of copies of each target nucleic
acid/adaptor library construct 308. These library constructs 308
are then cleaved 309 (for example, by restriction with a Type IIs
restriction endonuclease recognizing one or more sites in the
adaptor and cutting in the target nucleic acid sequence), and the
cycle continues to add second, third, fourth or more adaptors.
[0055] FIG. 4 is a schematic illustration of one aspect of a DNA
array 400 employing multi-adaptor nucleic acid library constructs.
The multi-adaptor nucleic acid library constructs in the form of
DNA nanoballs (DNBs) are seen at 402. DNBs are arrayed on a planar
matrix 404 having discrete sites 406. The DNBs 402 may be fixed to
the discrete sites by a variety of techniques, including covalent
attachment and non-covalent attachment. In one embodiment, the
surface of the matrix 406 may comprise attached capture
oligonucleotides that form complexes, e.g., double-stranded
duplexes, with a segment of an adaptor component of the DNB. In
other embodiments, capture oligonucleotides may comprise
oligonucleotide clamps, or like structures, that form triplexes
with adaptor oligonucleotides (see, e.g., U.S. Pat. No. 5,473,060).
In another embodiment, the surface of the array matrix 406 may have
reactive functionalities that react with complementary
functionalities on the DNBs to form a covalent linkage (see, e.g.,
Beaucage (2001), Current Medicinal Chemistry 8:1213-1244). Once the
DNBs are arrayed, the adaptors interspersed in the target nucleic
acids are used to acquire sequence information of the target
nucleic acids. A variety of sequencing methodologies may be used
with multi-adaptor nucleic acid library constructs, including but
not limited to hybridization methods as disclosed in U.S. Pat. Nos.
6,864,052; 6,309,824; 6,401,267; sequencing-by-synthesis methods as
disclosed in U.S. Pat. Nos. 6,210,891; 6,828,100, 6,833,246;
6,911,345; Margulies, et al. (2005), Nature 437:376-380 and
Ronaghi, et al. (1996), Anal. Biochem. 242:84-89; and
ligation-based methods as disclosed in U.S. Pat. No. 6,306,597; and
Shendure et al. (2005) Science 309:1728-1739, all of which are
incorporated by reference in their entirety.
[0056] In one aspect, the DNBs described herein--particularly those
with inserted and interspersed adapters--are used in sequencing by
combinatorial probe-anchor ligation reaction (cPAL) (see U.S. Ser.
No. 11/679,124, filed Feb. 24, 2007). In brief, cPAL comprises
cycling of the following steps: First, an anchor is hybridized to a
first adaptor in the DNBs (typically immediately at the 5' or 3'
end of one of the adaptors). Enzymatic ligation reactions are then
performed with the anchor to a fully degenerate probe population
of, e.g., 8-mer probes that are labeled, e.g., with fluorescent
dyes. Probes may have a length, e.g., about 6-20 bases, or,
preferably, about 7-12 bases. At any given cycle, the population of
8-mer probes that is used is structured such that the identity of
one or more of its positions is correlated with the identity of the
fluorophore attached to that 8-mer probe. For example, when 7-mer
sequencing probes are employed, a set of fluorophore-labeled probes
for identifying a base immediately adjacent to an interspersed
adaptor may have the following structure: 3'-F1-NNNNNNAp,
3'-F2-NNNNNNGp. 3'-F3- NNNNNNCp and 3'-F4-NNNNNNTp (where "p" is a
phosphate available for ligation). In yet another example, a set of
fluorophore-labeled 7-mer probes for identifying a base three bases
into a target nucleic acid from an interspersed adaptor may have
the following structure: 3'-F1-NNNNANNp, 3'-F2-NNNNGNNp.
3'-F3-NNNNCNNp and 3'-F4-NNNNTNNp. To the extent that the ligase
discriminates for complementarity at that queried position, the
fluorescent signal provides the identity of that base.
[0057] After performing the ligation and four-color imaging, the
anchor:8-mer probe complexes are stripped and a new cycle is begun.
With T4 DNA ligase, accurate sequence information can be obtained
as far as six bases or more from the ligation junction, allowing
access to at least 12 bp per adaptor (six bases from both the 5'
and 3' ends), for a total of 48 bp per 4-adaptor DNB, 60 bp per
5-adaptor DNB and so on.
[0058] FIG. 5 is a schematic illustration of the components that
may be used in an exemplary sequencing-by-ligation technique. A
construct 500 is shown with a stretch of target nucleic acid to be
analyzed interspersed with three adaptors, with the 5' end of the
stretch shown at 502 and the 3' end shown at 504. The target
nucleic acid portions are shown at 506 and 508, with adaptor 1
shown at 501, adaptor 2 shown at 503 and adaptor 3 shown at 505.
Four anchors are shown: anchor A1 (510), which binds to the 3' end
of adaptor 1 (501) and is used to sequence the 5' end of target
nucleic acid 506; anchor A2 (512), which binds to the 5' end of
adaptor 2 (503) and is used to sequence the 3' end of target
nucleic acid 506; anchor A3 (514), which binds to the 3' end of
adaptor 2 (503) and is used to sequence the 5' end of target
nucleic acid 508; and anchor A4 (516), which binds to the 5' end of
adaptor 3 (505) and is used to sequence the 3' end of target
nucleic acid 508.
[0059] Depending on which position that a given cycle is aiming to
interrogate, the 8-mer probes are structured differently.
Specifically, a single position within each 8-mer probe is
correlated with the identity of the fluorophore with which it is
labeled. Additionally, the fluorophore molecule is attached to the
opposite end of the 8-mer probe relative to the end targeted to the
ligation junction. For example, in the graphic shown here, the
anchor 530 is hybridized such that its 3' end is adjacent to the
target nucleic acid. To query a position five bases into the target
nucleic acid, a population of degenerate 8-mer probes shown here at
518 may be used. The query position is shown at 532. In this case,
this correlates with the fifth nucleic acid from the 5' end of the
8-mer probe, which is the end of the 8-mer probe that will ligate
to the anchor. In the aspect shown in FIG. 5, the 8-mer probes are
individually labeled with one of four fluorophores, where Cy5 is
correlated with A (522), Cy3 is correlated with G (524), Texas Red
is correlated with C (526), and FITC is correlated with T
(528).
[0060] Many different variations of cPAL or other
sequencing-by-ligation approaches may be selected depending on
various factors such as the volume of sequencing desired, the type
of labels employed, the number of different adaptors used within
each library construct, the number of bases being queried per
cycle, how the DNBs are attached to the surface of the array, the
desired speed of sequencing operations, signal detection approaches
and the like. In the aspect shown in FIG. 5 and described herein,
four fluorophores were used and a single base was queried per
cycle. It should, however, be recognized that eight or sixteen
fluorophores or more may be used per cycle, increasing the number
of bases that can be identified during any one cycle. The
degenerate probes (in FIG. 5, 8-mer probes) can be labeled in a
variety of ways, including the direct or indirect attachment of
radioactive moieties, fluorescent moieties, colorimetric moieties,
chemiluminescent moieties, and the like. Many comprehensive reviews
of methodologies for labeling DNA and constructing DNA adaptors
provide guidance applicable to constructing oligonucleotide probes
of the present invention. Such reviews include Kricka (2002), Ann.
Clin. Biochem., 39: 114-129; and Haugland (2006), Handbook of
Fluorescent Probes and Research Chemicals, 10th Ed.
(Invitrogen/Molecular Probes, Inc., Eugene); Keller and Manak
(1993), DNA Probes, 2nd Ed. (Stockton Press, New York, 1993); and
Eckstein (1991), Ed., Oligonucleotides and Analogues: A Practical
Approach (IRL Press, Oxford); and the like.
[0061] In one aspect, one or more fluorescent dyes are used as
labels for the oligonucleotide probes. Labeling can also be carried
out with quantum dots, as disclosed in the following patents and
patent publications, incorporated herein by reference: U.S. Pat.
Nos. 6,322,901; 6,576,291; 6,423,551; 6,251,303; 6,319,426;
6,426,513; 6,444,143; 5,990,479; 6,207,392; 2002/0045045;
2003/0017264; and the like. Commercially available fluorescent
nucleotide analogues readily incorporated into the degenerate
probes include, for example, Cascade Blue, Cascade Yellow, Dansyl,
lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green
514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red,
tetramethylrhodamine, Texas Red, the Cy fluorophores, the Alexa
Fluor.RTM. fluorophores, the BODIPY.RTM. fluorophores and the like.
FRET tandem fluorophores may also be used. Other suitable labels
for detection oligonucleotides may include fluorescein (FAM),
digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine
(BrdU), hexahistidine (6.times.His), phosphor-amino acids (e.g.
P-tyr, P-ser, P-thr) or any other suitable label.
[0062] Imaging acquisition may be performed by methods known in the
art, such as use of the commercial imaging package Metamorph. Data
extraction may be performed by a series of binaries written in,
e.g., C/C++, and base-calling and read-mapping may be performed by
a series of Matlab and Perl scripts. As described above, for each
base in a target nucleic acid to be queried (for example, for 12
bases, reading 6 bases in from both the 5' and 3' ends of each
target nucleic acid portion of each DNB), a hybridization reaction,
a ligation reaction, imaging and a primer stripping reaction is
performed. To determine the identity of each DNB in an array at a
given position, after performing the biological sequencing
reactions, each field of view ("frame") is imaged with four
different wavelengths corresponding to the four fluorescent, e.g.,
8-mers used. All images from each cycle are saved in a cycle
directory, where the number of images is 4.times. the number of
frames (for example, if a four-fluorophore technique is employed).
Cycle image data may then be saved into a directory structure
organized for downstream processing.
[0063] Data extraction typically requires two types of image data:
bright field images to demarcate the positions of all DNBs in the
array; and sets of fluorescence images acquired during each
sequencing cycle. The data extraction software identifies all
objects with the brightfield images, then for each such object,
computes an average fluorescence value for each sequencing cycle.
For any given cycle, there are four data-points, corresponding to
the four images taken at different wavelengths to query whether
that base is an A, G, C or T. These raw base-calls are
consolidated, yielding a discontinuous sequencing read for each
DNB. The next task is to match these sequencing reads against a
reference genome.
[0064] Information regarding the reference genome may be stored in
a reference table. A reference table may be compiled using existing
sequencing data on the organism of choice. For example human genome
data can be accessed through the National Center for Biotechnology
Information at ftp.ncbi.nih.gov/refseq/release, or through the J.
Craig Venter Institute at http://www.jcvi,org/researchhuref/. All
or a subset of human genome information can be used to create a
reference table for particular sequencing queries. In addition,
specific reference tables can be constructed from empirical data
derived from specific populations, including genetic sequence from
humans with specific ethnicities, geographic heritage, religious or
culturally-defined populations, as the variation within the human
genome may slant the reference data depending upon the origin of
the information contained therein.
[0065] In an alternative aspect of the claimed invention, parallel
sequencing of the target nucleic acids in the DNBs on a random
array is performed by combinatorial sequencing-by-hybridization
(cSBH), as disclosed by Drmanac in U.S. Pat. Nos. 6,864,052;
6,309,824; and 6,401,267. In one aspect, first and second sets of
oligonucleotide probes are provided, where each set has member
probes that comprise oligonucleotides having every possible
sequence for the defined length of probes in the set. For example,
if a set contains probes of length six, then it contains 4096
(4.sup.6) probes. In another aspect, first and second sets of
oligonucleotide probes comprise probes having selected nucleotide
sequences designed to detect selected sets of target
polynucleotides. Sequences are determined by hybridizing one probe
or pool of probes, hybridizing a second probe or a second pool or
probes, ligating probes that form perfectly matched duplexes on
their target nucleic acids, identifying those probes that are
ligated to obtain sequence information about the target nucleic
acid sequence, repeating the steps until all the probes or pools of
probes have been hybridized, and determining the nucleotide
sequence of the target nucleic acid from the sequence information
accumulated during the hybridization and identification
processes.
[0066] In yet another alternative aspect, parallel sequencing of
the target nucleic acids in the DNBs is performed by
sequencing-by-synthesis techniques as described in U.S. Pat. Nos.
6,210,891; 6,828,100, 6,833,246; 6,911,345; Margulies, et al.
(2005), Nature 437:376-380 and Ronaghi, et al. (1996), Anal.
Biochem. 242:84-89. Briefly, modified pyrosequencing, in which
nucleotide incorporation is detected by the release of an inorganic
pyrophosphate and the generation of photons, is performed on the
DNBs in the array using sequences in the adaptors for binding of
the primers that are extended in the synthesis.
Adaptor Insertion and Structure
[0067] The inability to control the orientation of adaptors with
respect to one another can have a number of undesired consequences.
The presence of adaptors in both orientations in a population of
target nucleic acid/adaptor library constructs requires the use of
two different anchor oligos in each sequencing reaction to enable
sequencing regardless of the orientation of a given adaptor. In
addition, sequencing of adaptors of unspecified orientation
requires either determination of the orientation of each
adaptor--adding at least one additional round of hybridization and
scanning to the sequencing process--or consideration of all
possible combinations of adaptor orientation during assembly of
sequencing reads from adaptors in the same target nucleic
acid/adaptor construct.
[0068] FIG. 6 is a schematic illustration of an insertion of a
second adaptor relative to a first adaptor in a nucleic acid
library construct. Again Process 600 begins with circular library
construct 602, having an inserted first adaptor 610. First adaptor
610 has a specific orientation, with a rectangle identifying the
"outer strand" of the first adaptor and a diamond identifying the
"inner strand" of the first adaptor (Ad1 orientation 610). A Type
IIs restriction endonuclease site in the first adaptor 610 is
indicated by the tail of arrow 601, and the site of cutting is
indicated by the arrow head. Process 603 comprises cutting with the
Type IIs restriction endonuclease, ligating first and second
adaptor arms of a second adaptor, and recircularization. As can be
seen in the resulting library constructs 604 and 606, the second
adaptor can be inserted in two different ways relative to the first
adaptor. In the desired orientation 604, the oval is inserted into
the circle's outer strand with the rectangle, and the bowtie is
inserted into the circle's inner strand with the diamond (Ad2
orientation 620). In the undesired orientation the oval is inserted
into the circle's inner strand with the diamond and the bowtie is
inserted into the circle's outer strand with the rectangle (Ad2
orientation 630).
[0069] FIG. 7 is a schematic representation of components of an
exemplary adaptor useful for selecting insertion orientation. A
basic schematic of an adaptor is shown at 700. The adaptor
comprises a 5' arm 701, a double-stranded region 702 and a 3' arm
703. Both the 5' and the 3' arms have a "T tail" 704 and a Type IIs
restriction endonuclease site 705 (here, EcoP15). The binding
region 702 is the region where the two arms of the adaptor come
together to be ligated in the circularization process (305 of FIG.
3). Structure 710 is the 5' arm of adaptor 700. Again, T tail 704
and the EcoP15 site 705 are shown, as well as the 5' arm 701 and
the binding region 712. Structure 720 is the 3' arm of adaptor 700.
Note the T tail 704 and the EcoP15 site 705, as well as the 3' arm
703 and the binding region 722. In the 5' arm, the binding region
712 is complementary to the binding region 722 of the 3' arm.
[0070] Because the aspects of the claimed invention work optimally
when library constructs are of a desired size and limited target
nucleic acid sequence, it is preferred that throughout the library
construction process the circularization reactions occur
intramolecularly. That is, that the separate constructs of the
library that are generated in the library construct assembly cycle
(as shown in FIG. 3) do not ligate to one another. Also, it is
preferred that only one set of adaptor arms for each adaptor used
in the library construction process be included per target nucleic
acid/adaptor construct. Thus, blocking oligos 717 and 727 are used
to block the binding regions 712 and 722 regions, respectively.
Blocker oligonucleotide 717 is complementary to binding sequence
716, and blocker olidonucleotide 727 is complementary to binding
sequence 726. In the schematic illustrations of the 5' adaptor arm
and the 3' adaptor arm, the underlined bases are ddC and the bolded
font bases are phosphorylated. Blocker oligonucleotides 717 and 727
are not covalently bound to the adaptor arms, and can be "melted
off" after ligation of the adaptor arms to the library construct
and before circularization; further, the dideoxy nucleotide (here,
ddC or alternatively a different non-ligatable nucleotide) prevents
ligation of blocker to adaptor. In addition or as an alternative,
in some aspects, the blocker oligo-adaptor arm hybrids contain a
one or more base gap between the adaptor arm and the blocker to
reduce ligation of blocker to adaptor. In some aspects, the
blocker/binding region hybrids have T.sub.ms of about 37.degree. C.
to enable easy melting of the blocker sequences prior to tail to
tail ligation (circularization).
[0071] Adaptor structure 730 is a schematic of the final adaptor,
where N is an unspecified base, a numeral "1" specifies bases added
to disrupt the palindrome (i.e., the EcoP15 site is flanked by A's
to isolate the 6-base palindrome formed by the EcoP15 sites on the
two arms of the adaptor), numeral "2" specifies bases that
correspond to the ddC in the blocker oligonucleotides, numeral "3"
specifies the EcoP15 site (CTGCTG) and numeral "4" specifies the T
bases designated for TA ligation to the A tailed target nucleic
acid. The adaptor shown as 900 and detailed at 930 would, in some
aspects, be appropriate for a first adaptor to be added in the
construction of a library. Adaptors added subsequently would, in
some aspects, have a single Type IIs restriction endonuclease site
rather than two sites, and, in some aspects, the Type IIs
restriction endonuclease sites in each adaptor would be different
from one another. Exemplary Type IIs restriction endonucleases
include, but are not limited to, Eco57M I, Mme I, Acu I, Bpm I,
BceA I, Bbv I, BciV I, BpuE I, BseM II, BseR I, Bsg I, BsmF I, BtgZ
I, Eci I, EcoP15 I, Eco57M I, Fok I, Hga I, Hph I, Mbo II, Mnl I,
SfaN I, TspDT I, TspDW I, Taq II, and the like.
[0072] In some aspects, the adaptors when assembled have a total
length of about 50 nucleotides. As shown above, in some aspects,
the adaptors are ligated to the target nucleic acid as two adaptor
arms, where each adaptor arm comprises two adaptor oligos (the two
complementary strands) and one blocker oligo. As shown the 5' ends
of all four adaptor arm oligos are phosphorylated to support
ligation to the insert and tail-to-tail ligation of 5' to 3'
adaptor arms. As shown, the 5' and 3' adaptor arms have 3'
overhangs at the adaptor-target nucleic acid ligation junctions, to
enable ligation to an A-tailed insert, and to suppress head-to-head
adaptor arm ligation. Also as shown, the 5' and 3' adaptor arms
have Type IIs restriction endonuclease recognition sites oriented
to enable cleavage of the adjacent target nucleic acid.
[0073] Again, the adaptor construct shown in FIG. 7 would be, in
some aspects, appropriate for a first adaptor to be inserted into a
library construct because it contains two Type IIs restriction
endonuclease recognition sites. Subsequently inserted adaptors
would, in some aspects, comprise a single Type IIs restriction
endonuclease recognition site oriented to enable cleavage of the
adjacent target nucleic acid. Additionally, in preferred aspects,
the 5' and 3' adaptor arms have anchor primer binding sites to
enable sequencing of adjacent target nucleic acids. The anchor
primer binding sites in some aspects overlap with the respective
Type IIs restriction endonuclease recognition site(s); however, in
other aspects the anchor primer binding sites do not overlap with
the Type IIs restriction endonuclease recognition site(s).
[0074] FIG. 8 is a schematic representation of adaptor insertion
allowing subsequent circularization of the target/adaptor
construct. The portion of the library construct seen in FIG. 8 is
adaptor-centric, showing target nucleic acid at 802 and 812, a 5'
adaptor arm at 804, a 5' adaptor arm blocking oligo at 806, a 3'
adaptor arm at 810, and a 3' adaptor arm blocking oligo at 808. The
T tail of the adaptor arms 804 and 810 and the A tail of the target
nucleic acids 802 and 812 are indicated. In process 801, the
adaptor arms are ligated to the target nucleic acid resulting in
target nucleic acid/5' adaptor arm structure 814, and target
nucleic acid/3' adaptor arm structure 816, with blocking oligos 806
and 808 still hybridized to the target nucleic acid/adaptor arm
structures. In process 803, the blockers are removed by melting,
and, in preferred aspects under dilute conditions to favor
intramolecular ligation of process 805. The resulting structure is
seen at 818. FIG. 8 illustrates the process of adaptor arm ligation
featuring blocking oliogs; however, other methods may be used to
block ligation-creating concatemers of adaptor arms or of library
constructs, including using adaptor arms that comprise a
restriction site, preferably a site for a restriction endonuclease
that cuts asymmetrically, such as Ava I. Alternatively, the adaptor
arms may comprise one or more uracil bases that can be selectively
cleaved using uracil-DNA glycosylase enzyme (Krokan et al, 1997)
with the resulting fragments then being melted off in the same way
the blocker oligo is melted off.
Selection of Adaptor Orientation by Selective Capture
[0075] The claimed methods provide selection of orientation by
selective capture (or "hybrid capture"), where a series of
purification steps is performed that result in the sequential
elimination of various undesired structures. A schematic of this
process is shown in FIG. 9. FIG. 9 shows the results of adding
first and second adaptor arms to a library construct that already
has a first adaptor interspersed within the target nucleic acid.
The first adaptor has a rectangle in the "top strand" and a diamond
in the "bottom strand." The second adaptor has an oval in the "top
strand" and a bowtie in the "bottom strand." The first arm of the
second adaptor is phosphorylated on the 5' end of the top strand
(designated by a "P"), whereas the second arm of the second adaptor
has a biotin (or other capturable functional moiety) on the 5' end
of the bottom strand (designated by a "B"). The functional moiety
may be part of the first or second arm of the second adaptor that
are ligated to the library construct; alternatively, after ligation
of the first and second arms of the second adaptor, the second
library constructs may be amplified with a functionalized primer
complementary to an end of one strand of the second adaptor, and
the functionalized amplified double-stranded second library
constructs are captured.
[0076] Ligation of the two arms of the second adaptor results in
four combinations: AB (with "P" strand 902 as a top strand and "B"
strand 904 as a bottom strand); AA (with "P" strands 902 as both a
top strand and a bottom strand; BB (with "B" strands 904 as both a
top strand and a bottom strand); and BA (with "B" strand 904 as a
top strand and "P" strand 902 as a bottom strand). Selective
capture of the biotinylated dsDNA (or otherwise functionalized
dsDNA) eliminates unbiotinylated AA structures in 906. Subsequent
denaturation and elution of unbiotinylated ssDNA from the captured
dsDNA eliminates double-biotinylated BB structures in 906. Next,
first adaptor top-strand containing ssDNAs (AB) are purified using
a first adaptor top-strand specific capture probe (a probe with a
sequence specific for a nucleotide sequence of the first-adaptor
top strand). The first adaptor top-strand ssDNA structure is then
circularized using, e.g., circLigase, and the resulting circles can
be amplified and converted to dsDNA using circle dependant
amplification.
EXAMPLES
[0077] A Tailing: Samples of 100 ng of fragmented genomic DNA were
prepared in Thermopol buffer, with dATP and Taq polymerase added.
The samples were then incubated at 70.degree. C. for 60 minutes and
cooled to 4.degree. C. The samples were then purified by Qiagen
MinElute columns.
[0078] Adaptor annealing: The A tailed fragmented genomic DNA
samples were mixed with T tailed adaptors and blocking oligos in a
buffer containing NaCl, Tris and EDTA. The samples were then heated
to 95.degree. C. for 5 minutes and then allowed to cool to room
temperature.
[0079] Adaptor ligation: The annealed adaptor/genomic DNA samples
were mixed with HB ligation buffer and T4 ligase. The samples were
then incubated at 14.degree. C. for two hours, 70.degree. C. for 10
minutes (to inactivate the T4 enzyme and remove the blocking
oligos) and cooled to 4.degree. C. The samples were then purified
by Qiagen MinElute columns.
[0080] Adaptor circularization: The linear fragmented genomic DNAs
now flanked by first and second arms of an adaptor were
circularized by incubation in epicenter buffer and T4 Ligase at
14.degree. C. for 14 hours. The samples were then heat inactivated
at 70.degree. C. for 10 minutes and then cooled to 4.degree. C.
[0081] The present specification provides a complete description of
the methodologies, systems and/or structures and uses thereof in
example aspects of the presently-described technology. Although
various aspects of this technology have been described above with a
certain degree of particularity, or with reference to one or more
individual aspects, those skilled in the art could make numerous
alterations to the disclosed aspects without departing from the
spirit or scope of the technology hereof. Since many aspects can be
made without departing from the spirit and scope of the presently
described technology, the appropriate scope resides in the claims
hereinafter appended. Other aspects are therefore contemplated.
Furthermore, it should be understood that any operations may be
performed in any order, unless explicitly claimed otherwise or a
specific order is inherently necessitated by the claim language. It
is intended that all matter contained in the above description and
shown in the accompanying drawings shall be interpreted as
illustrative only of particular aspects and are not limiting to the
embodiments shown. Changes in detail or structure may be made
without departing from the basic elements of the present technology
as defined in the following claims.
Sequence CWU 1
1
9115DNAArtificial SequenceSynthetic oligonucleotide 1acucuagcug
acuag 15235DNAArtificial SequenceTarget nucleic acid 2gagtnnnnnn
nnnnnnnnnn tgagatcgac tgatc 35330DNAArtificial SequenceSynthetic
oligonucleotide 3actgctgacg cttacgatgc acgatacgtc
30432DNAArtificial SequenceSynthetic oligonucleotide 4ttgacgactg
cgaatgctac gtgctatgca gt 32532DNAArtificial SequenceSynthetic
oligonucleotide 5tgcacgatac gtctacgatg cgaacagcag at
32630DNAArtificial SequenceSynthetic oligonucleotide 6cgtgctatgc
agatgctacg cttgtcgtct 30750DNAArtificial SequenceSynthetic
oligonucleotide 7aactgctgan nnnnnnnnng nnnnnnnnnn cnnnnnnnnn
nacagcagat 50849DNAArtificial SequenceSynthetic oligonucleotide
8aactgctgac gcttacgatg cacgatacgt ctacgatgcg aacagcaga
49949DNAArtificial SequenceSynthetic oligonucleotide 9tgacgactgc
gaatgctacg tgctatgcag atgctacgct tgtcgtcta 49
* * * * *
References