WO2013010062A2 - Réduction de la complexité d'acide nucléique - Google Patents

Réduction de la complexité d'acide nucléique Download PDF

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WO2013010062A2
WO2013010062A2 PCT/US2012/046624 US2012046624W WO2013010062A2 WO 2013010062 A2 WO2013010062 A2 WO 2013010062A2 US 2012046624 W US2012046624 W US 2012046624W WO 2013010062 A2 WO2013010062 A2 WO 2013010062A2
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Prior art keywords
nucleic acid
capture
adaptor
blocker
target polynucleotides
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PCT/US2012/046624
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English (en)
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WO2013010062A3 (fr
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Meredith GAVIN
Christopher CLOUSER
Tanya Sokolsky
Kimberly MATHER
Kevin Mckernan
Marie Callahan
Gary Bee
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Life Technologies Corporation
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Priority to US14/232,572 priority Critical patent/US20140243232A1/en
Publication of WO2013010062A2 publication Critical patent/WO2013010062A2/fr
Publication of WO2013010062A3 publication Critical patent/WO2013010062A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6832—Enhancement of hybridisation reaction

Definitions

  • the present teachings provide compositions, systems, methods and kits for reducing the complexity of nucleotide sequences in a nucleic acid sample comprising hybridizing a nucleic acid sample with one or more blocker oligonucleotides, and optionally with one or more nucleic acid capture oligonucleotides, to form a capture duplex.
  • Reducing the complexity of a nucleic acid sample produces a sub-population that is enriched for nucleic acids having desired sequences or lacking nucleic acids having undesirable sequences.
  • Complexity reducing methods typically employ sequence-specific hybridization to capture nucleic acids from a source such as genomic DNA, DNA libraries or RNA. The resulting enriched population can be used as probes, or can be quantitated or sequenced.
  • the present teachings provide compositions, systems, methods and kits for reducing the complexity of nucleotide sequences in a nucleic acid sample.
  • methods for reducing the complexity of nucleotide sequences in a nucleic acid sample comprise capturing target polynucleotides from an initial nucleic acid sample to obtain a nucleic acid subpopulation having desired sequences or lacking certain sequences.
  • an initial nucleic acid sample includes target polynucleotides and non-target polynucleotides.
  • each of the target polynucleotides and non-target polynucleotides can be joined to at least one nucleic acid adaptor to form target and non-target polynucleotide constructs, respectively.
  • methods for capturing target polynucleotides comprise contacting an initial nucleic acid sample with an oligonucleotide under conditions suitable to hybridize the oligonucleotide to a nucleic acid adaptor.
  • the oligonucleotide can be a blocker oligonucleotide which hybridizes to the nucleic acid adaptor, thereby reducing or blocking non-specific hybridization between a nucleic acid adaptor and other nucleic acids in the hybridization reaction.
  • the methods further comprise contacting the initial nucleic acid sample with a capture oligonucleotide.
  • a capture oligonucleotide hybridizes to at least a portion of the target polynucleotide. In some embodiments, the capture oligonucleotide hybridizes to at least a portion of the target polynucleotide to form a capture duplex.
  • the capture duplexes can be separated from the non-target polynucleotides that do not form duplexes to enrich target polynucleotides.
  • the methods can further comprise sequencing a target
  • methods for capturing target polynucleotides comprises: (a) providing a nucleic acid sample having a plurality of non-target polynucleotide constructs which include a plurality of non-target polynucleotides each joined to at least one nucleic acid adaptor, and the nucleic acid sample having a plurality of target polynucleotide constructs which include a plurality of target polynucleotides each joined to at least one nucleic acid adaptor; (b) contacting the nucleic acid sample with at least one blocker oligonucleotide which hybridizes with the at least one nucleic acid adaptor.
  • the method further comprises contacting the nucleic acid sample with at least one capture oligonucleotide which hybridizes to at least a portion of the plurality of target polynucleotides to form at least one capture duplex.
  • the methods further comprises separating the at least one capture duplex from non-target polynucleotides that do not form capture duplexes to enrich target polynucleotides.
  • the methods further comprise sequencing a target polynucleotide that formed a capture duplex.
  • methods for capturing target polynucleotides comprises: (a) providing a nucleic acid sample having a plurality of single- stranded non-target polynucleotide constructs which include a plurality of non-target polynucleotides each joined to at least one nucleic acid adaptor, and the nucleic acid sample having a plurality of single- stranded target polynucleotide constructs which include a plurality of target polynucleotides each joined to at least one nucleic acid adaptor; (b) contacting the nucleic acid sample with at least one blocker oligonucleotide which hybridizes with the at least one nucleic acid adaptor.
  • the methods further comprise contacting the nucleic acid sample with at least one capture oligonucleotide which hybridizes to at least a portion of the plurality of single-stranded target polynucleotides to form at least one capture duplex.
  • the methods further comprises: separating the at least one capture duplex from non-target polynucleotides that do not form capture duplexes to enrich target polynucleotides.
  • the methods further comprise sequencing a target polynucleotide that formed a capture duplex.
  • methods for capturing target polynucleotides comprises: (a) providing a nucleic acid sample having a plurality of single- stranded non-target polynucleotide constructs which include a plurality of non-target polynucleotides each joined to a first and a second nucleic acid adaptor, and the nucleic acid sample having a plurality of single- stranded target polynucleotide constructs which include a plurality of target polynucleotides each joined to a first and a second nucleic acid adaptor; (b) contacting the nucleic acid sample with a first blocker oligonucleotide which hybridizes with the first nucleic acid adaptor; (c) contacting the nucleic acid sample with a second blocker oligonucleotide which hybridizes with the second nucleic acid adaptor.
  • the method further comprises contacting the nucleic acid sample with at least one capture oligonucleotide which hybridizes to at least a portion of the plurality of single-stranded target polynucleotides to form at least one capture duplex.
  • the methods further comprises: separating the at least one capture duplex from non- target polynucleotides that do not form capture duplexes to enrich target polynucleotides.
  • the methods further comprise sequencing a target polynucleotide that formed a capture duplex.
  • methods for capturing target polynucleotides comprises: (a) providing a nucleic acid sample having a plurality of double-stranded non-target polynucleotide constructs which include a plurality of non-target polynucleotides each joined to at least one nucleic acid adaptor, and the nucleic acid sample having a plurality of double- stranded target polynucleotide constructs which include a plurality of target polynucleotides each joined to at least one nucleic acid adaptor; (b) denaturing the nucleic acid sample to generate a single- stranded nucleic acid sample having a plurality of single- stranded non-target polynucleotide constructs and a plurality of single-stranded target polynucleotide constructs; (c) hybridizing the single-stranded nucleic acid sample to at least one blocker oligonucleotide which hybridizes to the at least
  • the method further comprises contacting the single-stranded nucleic acid sample with at least one capture oligonucleotide which hybridizes to at least a portion of the single-stranded target polynucleotide to produce a plurality of capture duplexes.
  • the methods further comprises: separating the at least one capture duplex from non-target polynucleotides that do not form capture duplexes to enrich target
  • the methods further comprise sequencing a target polynucleotide that formed a capture duplex.
  • methods for capturing target polynucleotides comprises: (a) providing a nucleic acid sample having a plurality of double-stranded non-target polynucleotide constructs which include a plurality of non-target polynucleotides each joined to a first and a second nucleic acid adaptor, and the nucleic acid sample having a plurality of double- stranded target polynucleotide constructs which include a plurality of target polynucleotides each joined to a first and a second nucleic acid adaptor; (b) denaturing the nucleic acid sample to generate a single-stranded nucleic acid sample having a plurality of single-stranded non-target
  • the method further comprises contacting the single-stranded nucleic acid sample with at least one capture oligonucleotide which hybridizes to at least a portion of the plurality of single-stranded target polynucleotides to form at least one capture duplex.
  • the methods further comprises: separating the at least one capture duplex from non- target polynucleotides that do not form capture duplexes to enrich target polynucleotides.
  • the methods further comprise sequencing a target polynucleotide that formed a capture duplex.
  • the present teachings provide a capture duplex produced by any method disclosed herein.
  • the methods further comprise separating at least one capture duplex from a plurality of non-target polynucleotide constructs.
  • the separated capture duplex can form an enriched target polynucleotide.
  • the present teachings provide an enriched target polynucleotides produced by any method disclosed herein.
  • the capture oligonucleotide comprises a binding moiety.
  • the binding moiety comprises biotin.
  • the method further comprises contacting the binding moiety to a binding partner moiety.
  • the binding partner moiety comprises avidin or streptavidin.
  • the binding partner moiety can be attached to a bead.
  • the bead can be magnetic or paramagnetic.
  • the capture oligonucleotide in the capture duplex, includes a binding moiety which binds a binding partner moiety (which is attached to a bead).
  • the methods further comprise removing the bead from non-target polynucleotides.
  • the bead can be removed from the non-target polynucleotides by contacting the bead (e.g., paramagnetic bead) to a magnetic source and separating the magnet- bead complex from the non-target polynucleotides to form separated capture duplexes.
  • the bead e.g., paramagnetic bead
  • the separated capture duplexes form a plurality of enriched target polynucleotides.
  • the present teachings provide an enriched target polynucleotide produced by any method disclose herein.
  • a target polynucleotide can be sequenced by any sequencing method, including sequencing-by-synthesis, ion-based sequencing involving the detection of sequencing byproducts using ISFETs, chemical degradation sequencing, ligation-based sequencing, hybridization sequencing, pyrophosphate detection sequencing, capillary electrophoresis, gel electrophoresis, next-generation, massively parallel sequencing platforms, sequencing platforms that detect hydrogen ions or other sequencing by-products, and single molecule sequencing platforms.
  • a nucleic acid adaptor (e.g., a first nucleic acid adaptor) comprises a PI adaptor sequence according to any one of SEQ ID NOS:3 or 5.
  • a nucleic acid adaptor (e.g., a second nucleic acid adaptor) comprises an A adaptor sequence according to any one of SEQ ID NOS: 140 or 141.
  • a blocker oligonucleotide (e.g., a first blocker oligonucleotide) comprises a sequence according to SEQ ID NO: l 12 or 143.
  • a blocker oligonucleotide e.g., a second blocker
  • oligonucleotide comprises a sequence according to SEQ ID NOS: 139 or 142.
  • a barcoded blocker oligonucleotide (e.g., a second blocker oligonucleotide) comprises a sequence according to any one of SEQ ID NOS: 144-239.
  • Figure 1 is a schematic depicting non-limiting embodiments of complexity reducing methods, and blocker oligonucleotides and capture oligonucleotides.
  • oligonucleotide synthesis Enzymatic reactions and purification techniques are performed according to manufacturer' s specifications or as commonly accomplished in the art or as described herein.
  • the techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000).
  • the nomenclatures utilized in connection with, and the laboratory procedures and techniques described herein are those well known and commonly used in the art.
  • hybridize and “hybridization” and “hybridizing” (and other related terms) can include hydrogen bonding between two different nucleic acids, or between two different regions of a nucleic acid, to form a duplex nucleic acid.
  • Hybridization can comprise Watson-Crick or Hoogstein binding to form a duplex nucleic acid.
  • the two different nucleic acids, or the two different regions of a nucleic acid may be complementary, or partially complementary.
  • the complementary base pairing can be the standard A-T or C-G base pairing, or can be other forms of base-pairing interactions.
  • Duplex nucleic acids can include mismatched nucleotides. Complementary nucleic acid strands need not hybridize with each other across their entire length.
  • the present teachings provide compositions, systems, methods and kits for reducing the complexity of nucleotide sequences in a nucleic acid sample.
  • the phrases "reducing the complexity of nucleotide sequences" and “reducing the complexity” and “reducing nucleic acid complexity” and “complexity reducing” and “reduced complexity nucleotide sequences” may be used interchangeably, and refer to capturing target polynucleotides from an initial nucleic acid sample to obtain a nucleic acid subpopulation having desired sequences or lacking certain sequences.
  • an initial nucleic acid sample includes non-target polynucleotide constructs and target polynucleotide constructs, where the target polynucleotide constructs can be selectively captured.
  • methods for capturing target polynucleotides comprise increasing the efficiency of sequence-specific capture using oligonucleotides that block nonspecific hybridization between the capture oligonucleotides and adaptor sequences in an initial nucleic acid sample.
  • blocker oligonucleotides can hybridize to at least a portion of a nucleic acid adaptor to decrease non-specific hybridization.
  • nonspecific hybridization includes, renaturation of double-stranded polynucleotide constructs, hybridization between different polynucleotide constructs, secondary structure formation (e.g., stem-loops), or hybridization between a capture oligonucleotide and a nucleic acid adaptor sequence.
  • capturing target polynucleotides comprises sequence- specific capture of polynucleotides of interest from a nucleic acid sample having a plurality of target and non-target polynucleotides. In some embodiments, capturing target polynucleotides comprises hybridizing polynucleotides of interest with one or more capture oligonucleotides.
  • a nucleic acid sample can include a plurality of polynucleotide constructs (both target and non-target) each having a polynucleotide joined to one or more adaptor sequences.
  • methods for capturing target polynucleotides comprise contacting an initial nucleic acid sample with one or more blocker oligonucleotides ( Figure 1).
  • an initial nucleic acid sample includes target polynucleotides and non- target polynucleotides.
  • each of the target polynucleotides and the non- target polynucleotides can be joined to at least one nucleic acid adaptor to form target and non- target polynucleotide constructs, respectively.
  • methods for capturing target polynucleotides comprise contacting an initial nucleic acid sample with at least one blocker oligonucleotide under conditions suitable to hybridize the blocker oligonucleotide to a nucleic acid adaptor ( Figure 1).
  • the methods further comprise contacting the initial nucleic acid sample with a capture oligonucleotide ( Figure 1).
  • a blocker oligonucleotide hybridizes to at least a portion of the nucleic acid adaptor.
  • a capture oligonucleotide hybridizes to at least a portion of the target
  • the capture oligonucleotide hybridizes to at least a portion of the target polynucleotide to form a capture duplex.
  • methods for capturing target polynucleotides can further comprise separating the capture duplexes from nucleic acids in the initial sample that are not part of a capture duplex to enrich a target polynucleotide.
  • the capture duplexes can be denatured to release single- stranded target polynucleotides.
  • the capture duplexes can be nucleotide sequences having reduced complexity.
  • the enriched target polynucleotides can be nucleotide sequences having reduced complexity.
  • the released single-stranded target polynucleotides can be nucleotide sequences having reduced complexity.
  • methods for capturing target polynucleotides comprise: (a) providing a nucleic acid sample having a plurality of non-target polynucleotide constructs which include a plurality of non-target polynucleotides each joined to at least one nucleic acid adaptor, and the nucleic acid sample having a plurality of target polynucleotide constructs which include a plurality of target polynucleotides each joined to at least one nucleic acid adaptor; (b) preventing non-specific hybridization by contacting the nucleic acid sample with at least one blocker oligonucleotide which hybridizes with the at least one nucleic acid adaptor.
  • the method further comprises contacting the nucleic acid sample with at least one capture oligonucleotide which hybridizes to at least a portion of the plurality of target polynucleotides to form at least one capture duplex. In some embodiments, the methods further comprises separating the at least one capture duplex from non-target polynucleotides that do not form capture duplexes to enrich target polynucleotides. In some embodiments, the methods further comprise sequencing a target polynucleotide that formed a capture duplex.
  • methods for capturing target polynucleotides comprise: (a) providing a nucleic acid sample having a plurality of single- stranded polynucleotides each joined to at least a first adaptor sequence, wherein the plurality of single- stranded polynucleotides includes non-target polynucleotide sequences and at least one target polynucleotide sequence; (b) hybridizing a plurality of a first blocker oligonucleotide to the nucleic acid sample under suitable hybridization conditions, wherein the first blocker oligonucleotide includes a nucleotide sequence that hybridizes to at least a portion of the first adaptor sequence; (c) hybridizing a plurality of a capture oligonucleotide to the nucleic acid sample under suitable hybridization conditions to produce a plurality of capture duplexes, wherein the capture oligonucleotides include a sequence that hybridizes to at
  • the methods further comprise: (d) separating the plurality of capture duplexes from the non-duplexed single- stranded polynucleotides by binding the binding moiety on the capture duplexes with a binding partner moiety.
  • the binding moiety comprises biotin.
  • the binding partner moiety comprises avidin or strep tavidin.
  • methods for capturing target polynucleotides comprise: (a) providing a nucleic acid sample having a plurality of single- stranded polynucleotides each joined to a first adaptor sequence and a second adaptor sequence, wherein the plurality of single- stranded polynucleotides includes non-target polynucleotide sequences at least one target polynucleotide sequence; (b) hybridizing a plurality of a first blocker oligonucleotide to the nucleic acid sample under suitable hybridization conditions, wherein the first blocker
  • oligonucleotide includes a nucleotide sequence that hybridizes to the first adaptor sequence; (b) hybridizing a plurality of a second blocker oligonucleotide to the nucleic acid sample under suitable hybridization conditions, wherein the second blocker oligonucleotide includes a nucleotide sequence that hybridizes to the second adaptor sequence; (c) hybridizing a plurality of a capture oligonucleotide to the nucleic acid sample under suitable hybridization conditions to produce a plurality of capture duplexes, wherein the capture oligonucleotides include a sequence that hybridizes to at least a portion of the target polynucleotide sequence.
  • the capture oligonucleotide includes a binding moiety.
  • the methods further comprise: (d) separating the plurality of capture duplexes from the non-duplexed single- stranded polynucleotides by binding the binding moiety on the capture duplexes with a binding partner moiety.
  • the binding moiety comprises biotin.
  • the binding partner moiety comprises avidin or streptavidin.
  • methods for capturing target polynucleotides comprise: (a) providing a nucleic acid sample having a plurality of double-stranded polynucleotides each joined to at least one adaptor sequence, wherein the plurality of double- stranded polynucleotides includes non-target polynucleotide sequences and at least one target polynucleotide sequence; (b) denaturing the double- stranded polynucleotides to generate a plurality of single- stranded polynucleotides each joined to at least one adaptor sequence; (c) hybridizing a plurality of a blocker oligonucleotide to the plurality of single- stranded polynucleotides under suitable hybridization conditions, wherein the blocker oligonucleotide includes a nucleotide sequence that hybridizes to the adaptor sequence; (d) hybridizing a plurality of a capture oligonucleo
  • the capture oligonucleotide includes a binding moiety.
  • the methods further comprise: (d) separating the plurality of capture duplexes from the non-duplexed single-stranded polynucleotides by binding the binding moiety on the capture duplexes with a binding partner moiety.
  • the binding moiety comprises biotin.
  • the binding partner moiety comprises avidin or streptavidin.
  • methods for capturing target polynucleotides comprise: (a) providing a nucleic acid sample having a plurality of double-stranded polynucleotides each joined to a first adaptor sequence and a second adaptor sequence, wherein the plurality of double-stranded polynucleotides includes non-target polynucleotide sequences at least one target polynucleotide sequence; (b) denaturing the double- stranded polynucleotides to generate a plurality of single-stranded polynucleotides each joined to a first adaptor sequence and a second adaptor sequence; (c) hybridizing a plurality of a first blocker oligonucleotide to the plurality of the single- stranded polynucleotides under suitable hybridization conditions, wherein the first blocker oligonucleotide includes a nucleotide sequence that hybridizes to the first adaptor sequence; (d) hybrid
  • the methods further comprise: (f) separating the plurality of capture duplexes from the non-duplexed single-stranded
  • polynucleotides by binding the binding moiety on the capture duplexes with a binding partner moiety.
  • the binding moiety comprises biotin.
  • the binding partner moiety comprises avidin or streptavidin.
  • a nucleic acid sample can include a plurality of polynucleotides having the same or different sequences.
  • a nucleic acid sample can comprise a plurality of target polynucleotides and/or non-target polynucleotides that are each joined to at least one nucleic acid adaptor to form target and non-target polynucleotide constructs which are part of a nucleic acid library (e.g., fragment libraries, barcoded fragment libraries, mate pair libraries and/or barcoded mate pair libraries).
  • a plurality of target polynucleotides and/or non-target polynucleotides can be joined to nucleic acid adaptors having the same or different sequences.
  • a nucleic acid sample can initially include double stranded nucleic acids that can be denatured to form a plurality of single stranded nucleic acids.
  • the single stranded nucleic acids can hybridize with the blocker oligonucleotides and/or hybridize with the capture oligonucleotides to form capture duplexes.
  • a nucleic acid sample can initially include single stranded nucleic acids that can hybridize with the blocker oligonucleotides and/or hybridize with the capture oligonucleotides to form capture duplexes.
  • hybridization reactions can be conducted in an aqueous or nonaqueous solution. In some embodiments, hybridization reactions can be conducted under stringent or less-than-stringent hybridization conditions. In some embodiments, a nucleic acid sample can be hybridized essentially simultaneously with a mixture or with the same type of capture oligonucleotides, and/or with a mixture or with the same type of blocker
  • a nucleic acid sample can be hybridized serially with one or more types of capture oligonucleotides or with one or more types of blocker oligonucleotides.
  • a combination of essentially simultaneous and/or serial hybridization modes can be used to hybridize a nucleic acid sample with capture oligonucleotides and/or with blocker oligonucleotides.
  • a nucleic acid sample can be hybridized to blocker oligonucleotides and/or to capture oligonucleotides in one round or multiple rounds of hybridization reactions.
  • double-stranded capture oligonucleotides and/or double- stranded blocker oligonucleotides can be denatured to become single-stranded for hybridization to the polynucleotide constructs.
  • capture oligonucleotides and/or blocker oligonucleotides can initially be single-stranded for hybridization to the polynucleotide constructs.
  • all steps of a target polynucleotide capture method can be conducted in one reaction vessel (e.g., a well) or different steps can be conducted in different reaction vessels (e.g., wells).
  • enriched target polynucleotides generated from separate capture reactions can be pooled together.
  • different initial nucleic acid samples can be pooled together and then subjected to capture reactions.
  • methods for capturing target polynucleotides can be conducted on any polynucleotide construct which is prepared for sequencing on any type of sequencing platform, including sequencing by oligonucleotide probe ligation and detection (e.g., SOLiDTM from Life Technologies, WO 2006/084131), probe-anchor ligation sequencing (e.g., Complete GenomicsTM or PolonatorTM), sequencing- by-synthesis (e.g., Genetic Analyzer and HiSeqTM, from niumina), pyrophosphate sequencing (e.g., Genome Sequencer FLX from 454 Life Sciences), ion-sensitive sequencing (e.g., Personal Genome Machine (PGMTM) and Ion ProtonTM Sequencer, both from Ion Torrent Systems, Inc.), and single molecule sequencing platforms (e.g., HeliScopeTM from HelicosTM).
  • sequencing by oligonucleotide probe ligation and detection e.g., SOLiDTM from Life Technologies,
  • the first adaptor sequence can include a PI sequence according to any one of SEQ ID NOS: l-5.
  • the second adaptor sequence can include a P2 sequence according to any one of SEQ ID NOS:6-12 or an A sequence according to any one of SEQ ID NOS: 131-134, 140 or 141.
  • the second adaptor sequence can include a barcoded sequence according to any one of SEQ ID NOS: 16-l l l.
  • the first blocker oligonucleotide can include a sequence that can hybridize to a PI adaptor.
  • a first blocker oligonucleotide can include a sequence according to SEQ ID NO: 112.
  • the second blocker oligonucleotide can include a sequence that can hybridize to an Ion A adaptor, an internal adaptor sequence, barcoded sequence or P2 adaptor sequence.
  • a second blocker oligonucleotide can include a sequence according to any of SEQ ID NO: 139 or SEQ ID NOS: 113-128.
  • methods for capturing target polynucleotides can further comprise separating the capture duplexes from nucleic acids in the sample that are not part of a capture duplex to enrich target polynucleotides.
  • a separating step can produce enriched target polynucleotides.
  • a separating step can be conducted with a paramagnetic bead separation reaction.
  • a capture oligonucleotide can be attached to a binding moiety (e.g., biotin).
  • a bead can be attached to a binding partner moiety (e.g., avidin or strep tavidin).
  • the binding moiety can bind the binding partner moiety.
  • a bead can be magnetic or paramagnetic.
  • a separating step can comprise binding a capture oligonucleotide (which is hybridized to a portion of a target polynucleotide construct and which is attached to a binding moiety) to a paramagnetic bead which is attached to a binding partner moiety to form a capture duplex -bead complex.
  • a paramagnetic bead which is attached to a binding partner moiety comprises a DynabeadsTM M-270 (from Invitrogen, Carlsbad, CA).
  • a separating step can further include removing the capture duplex -bead complex from non-target polynucleotide constructs that do not form a duplex.
  • the removing step can employ a magnetic source to attract the paramagnetic bead, to separate the capture duplex-bead complex from the non-duplexes, to produce enriched target polynucleotides.
  • additional steps can include a washing step, for example to remove: unhybridized blocker oligonucleotides, unhybridized capture oligonucleotides and/or unhybridized polynucleotides.
  • enriched target polynucleotides can be nucleotide sequences having reduced complexity.
  • methods for capturing target polynucleotides can further comprise releasing enriched target polynucleotides.
  • releasing enriched target polynucleotides can include denaturing a capture duplex to release the target polynucleotide from the capture oligonucleotide, thereby producing a released target polynucleotide.
  • denaturing can include methods well known in the art for nucleic acid melting, such as employing any combination of elevated temperatures, decreased salt concentrations (e.g., sodium), and/or increased formamide concentrations.
  • nucleic acid melting conditions employ temperatures above the melting temperature for a given nucleic acid length and %GC content.
  • released target polynucleotides can be nucleotide sequences having reduced complexity.
  • target polynucleotides are not released from a capture duplex.
  • target polynucleotides remain hybridized to at least one capture oligonucleotide (bound or unbound to a bead).
  • conditions that are suitable for nucleic acid hybridization and/or washing conditions include parameters such as salts, buffers, pH, temperature, GC% content of the capture oligonucleotides, GC% content of the blocker oligonucleotides, GC% content of the target polynucleotide, and/or time.
  • conditions suitable for hybridizing or washing polynucleotides with capture oligonucleotides and/or with blocker oligonucleotides can include hybridization solutions having sodium salts, such as NaCl, sodium citrate and/or sodium phosphate.
  • hybridization or wash solutions can include about 10-75% formamide and/or about 0.01-0.7% sodium dodecyl sulfate (SDS).
  • a hybridization solution can be a stringent hybridization solution which can include any combination of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, 0.1% SDS, and/or 10% dextran sulfate.
  • the hybridization or washing solution can include any combination of non-specific competitor nucleic acids such as human Cot-1 DNA and/or salmon sperm DNA.
  • the hybridization or washing solution can include BSA (bovine serum albumin).
  • BSA bovine serum albumin
  • hybridization or washing can be conducted at a temperature range of about 25-90° C, or about 30-75° C, or about 40-60° C, or about 60-80° C, or about 80- 99° C, or higher.
  • hybridization or washing can be conducted for a time range of about 1-6 hours, or about 6-12 hours, or about 12-24 hours, or about 24-36 hours, or about 36-48 hours, or about 2-3 days, or about 3-4 days, or about 4-5 days, or about 5-6 days, or about 6-7 days, or about 7-8 days, or more than 8 days.
  • hybridization or wash conditions can be conducted at a pH range of about 5-10, or about pH 6-9, or about pH 6.5-8, or about pH 6.5-7.
  • capture oligonucleotides and/or blocker oligonucleotides can be reacted with an amount of single- stranded or double- stranded polynucleotide constructs (e.g., nucleic acid library) of about 100-250 ng, or about 250-500 ng, or about 500-650 ng, or about 650-800 ng, or about 800-1000 ng, or more.
  • nucleic acid library e.g., nucleic acid library
  • thermal melting temperature (T m ) for nucleic acids can be a temperature at which half of the nucleic acid strands are double- stranded and half are single- stranded under a defined condition.
  • a defined condition can include ionic strength and pH in an aqueous reaction condition.
  • a defined condition can be modulated by altering the concentration of salts (e.g., sodium), temperature, pH, buffers, and/or formamide.
  • the calculated thermal melting temperature can be at about 5-30° C below the T m , or about 5-25° C below the T m , or about 5-20° C below the T m , or about 5-15° C below the T m , or about 5-10° C below the T m .
  • Methods for calculating a T m are well known and can be found in Sambrook (1989 in "Molecular Cloning: A Laboratory Manual", 2 nd edition, volumes 1-3; Wetmur 1966, J. Mol. Biol., 31:349-370; Wetmur 1991 Critical Reviews in Biochemistry and Molecular Biology, 26:227-259).
  • Other sources for calculating a T m for hybridizing or denaturing nucleic acids include OligoAnalyze (from Integrated DNA Technologies) and Primer3 (distributed by the Whitehead Institute for Biomedical Research).
  • the present teachings provide a blocker oligonucleotide comprising an oligonucleotide that can hybridize to at least a portion of a nucleic acid adaptor.
  • a blocker oligonucleotide can be a nucleic acid, including double- stranded, single- stranded, DNA, RNA or DNA/RNA hybrid.
  • a blocker oligonucleotide comprises a sequence having full or partial complementarity with a nucleic acid adaptor.
  • the sequence and length of a blocker oligonucleotide can be designed based on the sequence and length of any nucleic acid adaptor.
  • nucleic acid adaptors include sequences according to any of SEQ ID NOS: 1-12, 15-111, 129-138, and 140, 141.
  • blocker oligonucleotides include sequences according to any of SEQ ID NOS: 112-128, 139 and 142-239.
  • the present teachings provide a capture duplex comprising a polynucleotide construct hybridized to at least one capture oligonucleotide.
  • a capture oligonucleotide can be a nucleic acid, including double- stranded, single- stranded, DNA, RNA or DNA/RNA hybrid.
  • a capture oligonucleotide comprises a sequence having full or partial complementarity with at least a portion of at least one target polynucleotide sequence.
  • the sequence and length of a capture oligonucleotide can be designed based on the sequence and length of any target polynucleotide sequence.
  • a capture duplex can also be hybridized to at least one blocker oligonucleotide.
  • the polynucleotide construct includes a polynucleotide joined to at least one nucleic acid adaptor.
  • the polynucleotide (which is joined to the at least one adaptor) can be a target polynucleotide.
  • the capture oligonucleotide can hybridize to at least a portion of the target polynucleotide.
  • the blocker oligonucleotide can hybridize to at least a portion of the nucleic acid adaptor.
  • a nucleic acid adaptor comprises a sequence according to any of SEQ ID NOS: 1-12, 15-111, 129-138, and 140, 141.
  • a blocker oligonucleotide comprises a sequence according to any of SEQ ID NOS: 112-128, 139 and 142- 239.
  • a capture duplex can be located among a population of duplexed and non-duplexed polynucleotide constructs.
  • a capture duplex can be generated by employing any method described herein or methods well known in the art.
  • the present teachings provide an enriched target polynucleotide comprising a capture duplex that is separated away from polynucleotide constructs that do not form duplexes.
  • a capture duplex comprises a polynucleotide construct hybridized to at least one capture oligonucleotide.
  • a capture duplex can also be hybridized to at least one blocker oligonucleotide.
  • an enriched target polynucleotide can be attained by conducting a paramagnetic bead separation reaction to separate a capture duplex from polynucleotide constructs that do not form duplexes with a capture oligonucleotide.
  • an enriched target polynucleotide can be washed to remove unhybridized blocker oligonucleotides, unhybridized capture oligonucleotides and/or unhybridized polynucleotides.
  • the capture oligonucleotide can be attached to a binding moiety.
  • the binding moiety can be biotin.
  • a bead can be attached to a binding partner moiety, wherein the binding partner moiety binds to a binding moiety.
  • the binding partner moiety can be avidin or streptavidin.
  • the bead can be magnetic or paramagnetic.
  • a population of capture duplexes and non-duplex polynucleotide constructs can be contacted with a bead which is attached to a binding partner moiety.
  • a capture duplex can be contacted with a bead which is attached to a binding partner moiety, wherein the binding partner moiety can bind the binding moiety on the capture polynucleotide (which is hybridized to the target polynucleotide).
  • a paramagnetic bead (which is attached to a binding partner moiety) can bind to a binding moiety.
  • an enriched target polynucleotide can be denatured to separate the target polynucleotide construct from the capture oligonucleotide, to produce a released target polynucleotide.
  • a denaturing step can be omitted so that a target polynucleotide construct remains hybridized to a capture oligonucleotide.
  • enriched target polynucleotides and released target polynucleotides can be generated by employing any method described herein or methods well known in the art.
  • blocker oligonucleotides comprising an oligonucleotide.
  • blocker oligonucleotides can be DNA, cDNA, RNA, RNA/DNA hybrids, or analogs thereof.
  • Blocker oligonucleotides can be single- stranded or double-stranded nucleic acids (or analogs thereof).
  • Blocker oligonucleotides can include one or more nucleotide or nucleoside analogs, such as locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • Blocker oligonucleotides can be any length, including about 5-10 bp, or about 10-20 bp, or about 20-30 bp, or about 30-40 bp, or about 40-50 bp, or about 50-60 bp, or about 60-70 bp, or about 70-80 bp, or about 80-90 bp, or about 90-100 bp, or longer.
  • blocker oligonucleotides can include degenerate bases. In some embodiments, blocker oligonucleotides can include one or more inosine residues.
  • blocker oligonucleotides can include at least one scissile linkage.
  • a scissile linkage can be susceptible to cleavage or degradation by an enzyme or chemical compound.
  • blocker oligonucleotides can include at least one phosphorothiolate, phosphorothioate, and/or phosphoramidate linkage.
  • blocker oligonucleotides can include nucleotide sequences that can hybridize to any portion of a polynucleotide construct.
  • a blocker for example, a blocker
  • blocker oligonucleotides can include nucleotide sequences that are fully complementary (e.g., base pairing A-T and/or C/G) or partially complementary (e.g., mis-match pairing A with C or G, T with C or G, C with A or T, or G with A or T) to any portion of the polynucleotides or polynucleotide constructs.
  • blocker oligonucleotides can include nucleotide sequences that are complementary to at least a portion of one or more adaptors (e.g., PI, P2, A, internal, barcoded or universal adaptors) or can include nucleotide sequences that are complementary to a sequencing primer or amplification primer sequence, for example a primer sequence selected from SEQ ID NOS: 112-128).
  • blocker oligonucleotides can include nucleotide sequences of at least a portion of one or more adaptors (e.g., PI, P2, A, internal, barcoded or universal adaptors) or a sequencing primer or amplification primer sequence.
  • blocker oligonucleotides can include nucleotide sequences selected from SEQ ID NOS: 112-128, 135-139 and 142-239. These can optionally be used in conjunction with adapters and/or primers including nucleotide sequences selected from SEQ ID NOS: 129-134.
  • blocker oligonucleotides can include nucleotide sequences that are complementary to any combination of one or more adaptors.
  • blocker oligonucleotides can include nucleotide sequences that are complementary to any one or any combination of adaptor sequences, including: PI; P2; A; internal adaptor; and/or any barcode adaptor (any of SEQ ID NOS: 16-111).
  • PI polypeptide sequence
  • P2 polypeptide
  • A internal adaptor
  • barcode adaptor any of SEQ ID NOS: 16-111
  • Blocker oligonucleotides can include nucleotide sequence that can hybridize to any adaptor sequence that can be used to construct any type of nucleic acid library, including adaptor sequences for: SOLiDTM library (from Life Technologies, WO 2006/084131), Complete
  • GenomicsTM library PolonatorTM library, Genetic Analyzer library (Illumina), HiSeqTM library (niumina), Genome Sequencer FLX library (454 Life Sciences), Personal Genome Machine library (Ion Torrent Systems, Inc.), Ion ProtonTM Sequencer (Ion Torrent Systems, Inc.) and HeliScopeTM library (HelicosTM).
  • compositions and methods for capturing target polynucleotides where polynucleotides can be DNA, RNA, chimeric
  • polynucleotides can be single-stranded or double-stranded nucleic acids.
  • polynucleotides can be isolated in any form including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified (e.g., PCR amplified), cDNA, RNA (e.g., precursor mRNA, mRNA, miRNA, miRNA binding sites, fRNA), oligonucleotide, or any type of nucleic acid library.
  • polynucleotides can be isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, and viruses; cells; tissues; normal or diseased cells or tissues or organs, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen; environmental samples; culture samples; or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods.
  • polynucleotides can be chemically synthesized to include any type of nucleic acid analog.
  • polynucleotides can be isolated from a formalin-fixed tissue, or from a paraffin-embedded tissue, or from a formalin-fix paraffin- embedded (FFPE) tissue.
  • FFPE formalin-fix paraffin- embedded
  • polynucleotides can be polynucleotide fragments which can be generated enzymatically, chemically, or using any type of physical force (e.g., sonication, nebulization, or cavitation).
  • polynucleotides can be enzymatically fragmented by reacting with a restriction endonuclease.
  • polynucleotides can be enzymatically fragmented by nicking and nick translating the nick (in the presence or absence of nucleic acid binding proteins) to generate double- stranded breaks using any method disclosed in application No. PCT/US2012/039691, filed May 25, 2012, or U.S. Serial No.
  • polynucleotides can be enzymatically fragmented by binding polynucleotides with histones and cleaving with a nuclease (U.S. Patent No. 8,202,691, issued June 19, 2012).
  • polynucleotide fragments can be about 100-200 bp, or about 200-250 bp, or about 250-300 bp, or about 300-400 bp, or about 400-500 bp, or about 500-1000 bp, or about 100 bp - 1000 bp, or about 1 kb - 50 kb, or about 50 kb - 100 kb, or about 100-250 kb, or about 250-500 kb, or about 500-750 kb, or about 750-1000 bp, or about 1000 bp to about 1 Mb, or about 1-10 Mb, or about 10-20 Mb, or about 20-30 Mb, or about 30- 40 Mb, or about 40-50 Mb, or longer.
  • methods for capturing target polynucleotides can be conducted with starting nucleic acid fragments in an amount of about 0.01-0.1 ng, or about 0.1-1 ng, or about 1-5 ng, or about 5-10 ng, or about 10-50 ng, or about 50-100 ng, or about 100-500 ng, or about 500-1000 ng, or about 1-2 ug, or about 2-5 ug, or about 5-10 ug, or about 10-50 ug, or about 50-100 ug, or about 100-500 ug, or about 500-1000ug, or more.
  • the present teachings provide compositions and methods for capturing target polynucleotides, where at least one end of polynucleotides can be joined to any combination of one or more nucleic acid adaptors to form a polynucleotide construct.
  • one or both ends of a polynucleotide can be joined to at least one nucleic acid adaptor to generate a polynucleotide construct (e.g., Figure 1).
  • one end of a polynucleotide can be joined to a first adaptor and the other end of the polynucleotide can be joined to a second adaptor.
  • the first and second adaptors can be the same or different adaptors.
  • Nucleic acid adaptors can include sequences: PI, P2, A, internal adaptor, barcoded sequences, amplification primer sequences, sequencing primer sequences, and complementary sequences thereof.
  • polynucleotides can be joined to a first adaptor (e.g., PI adaptor) and a second adaptor (e.g., A, internal adaptor, barcode and/or P2 adaptors ) ( Figure 1).
  • polynucleotides and adaptors can be joined by ligation.
  • a polynucleotide can be joined to an adaptor with a ligase enzyme.
  • a polynucleotide can be joined to an adaptor by annealing or by conducting a primer extension reaction.
  • the length of polynucleotide constructs can be about 100-200 bp, or about 200-250 bp, or about 250-300 bp, or about 300-400 bp, or about 400-500 bp, or about 500-1000 bp, or about 100 bp - 1000 bp, or longer.
  • the present teachings provide compositions and methods for capturing target polynucleotides, where a polynucleotide can be joined to one or more nucleic acid adaptors.
  • a nucleic acid adaptor e.g., a first and/or second adaptor
  • an adaptor can include one or more ribonucleoside residues.
  • an adaptor can be single- stranded or double- stranded nucleic acids, or can include single-stranded and/or double-stranded portions.
  • an adaptor can have any structure, including linear, hairpin, forked, or stem-loop.
  • an adaptor can be a blocking oligonucleotide adaptor which comprises a double- stranded oligonucleotide adaptor (duplex) having an overhang cohesive portion.
  • oligonucleotide adaptors can hybridize with each other.
  • each end of a polynucleotide can be joined to a blocker oligonucleotide and the cohesive portions can be hybridized to each other to generate a circular nucleic acid molecule.
  • an adaptor can have any length, including fewer than 10 bases in length, or about 10-20 bases in length, or about 20-50 bases in length, or about 50-100 bases in length, or longer.
  • an adaptor can have any combination of blunt end(s) and/or sticky end(s).
  • at least one end of an adaptor can be compatible with at least one end of a nucleic acid fragment.
  • a compatible end of an adaptor can be joined to a compatible end of a nucleic acid fragment.
  • an adaptor can have a 5' or 3' overhang end.
  • an adaptor can include a monomeric sequence (e.g., AAA, TTT, CCC, or GGG) of any length, or an adaptor can include a complex sequence (e.g., non- monomeric sequence), or can include both monomeric and complex sequences.
  • an adaptor can have a 5' or 3' tail.
  • the tail can be one, two, three, or more nucleotides in length.
  • an adaptor can have a tail comprising A, T, C, G and/or U.
  • an adaptor can have a monomeric tail sequence of any length.
  • at least one end of an adaptor can have a tail that is compatible with a tail on one end of a nucleic acid fragment.
  • an adaptor can include an internal nick.
  • an adaptor can have at least one strand that lacks a terminal 5' phosphate residue.
  • an adaptor lacking a terminal 5' phosphate residue can be joined to a nucleic acid fragment to introduce a nick at the junction between the adaptor and the nucleic acid fragment.
  • an adaptor can include a nucleotide sequence that is part of, or is complementary to, a PI sequence (SEQ ID NOS: l-5), P2 sequence (SEQ ID NOS:6-12), A adaptor sequence (SE Q ID NOS: 140-141), internal adaptor, barcode sequence (SEQ ID NOS: l-5), P2 sequence (SEQ ID NOS:6-12), A adaptor sequence (SE Q ID NOS: 140-141), internal adaptor, barcode sequence (SEQ ID NOS: l-5), P2 sequence (SEQ ID NOS:6-12), A adaptor sequence (SE Q ID NOS: 140-141), internal adaptor, barcode sequence (SEQ ID NOS: l-5), P2 sequence (SEQ ID NOS:6-12), A adaptor sequence (SE Q ID NOS: 140-141), internal adaptor, barcode sequence (SEQ ID NOS: l-5), P2 sequence (SEQ ID NOS:6-12), A adaptor sequence (SE Q ID NOS: 140-141), internal adaptor
  • an adaptor can include degenerate sequences.
  • an adaptor can include one or more inosine residues.
  • a barcode adaptor can include a uniquely identifiable sequence.
  • a barcode adaptor can be used for constructing multiplex nucleic acid libraries.
  • an adaptor can include at least one scissile linkage.
  • a scissile linkage can be susceptible to cleavage or degradation by an enzyme or chemical compound.
  • an adaptor can include at least one
  • an adaptor can include identification sequences.
  • an identification sequences can be used for sorting or tracking.
  • an identification sequences can be a unique sequence (e.g., barcode sequence).
  • a barcode sequence can allow identification of a particular adaptor among a mixture of different adaptors having different barcodes sequences.
  • a mixture can include 2, 3, 4, 5, 6, 7-10, 10-50, 50-100, 100-200, 200-500, 500-1000, or more different adaptors having unique barcode sequences.
  • an adaptor can include any type of restriction enzyme recognition sequence, including type I, type II, type lis, type IIB, type III or type IV restriction enzyme recognition sequences.
  • an adaptor can include a cell regulation sequences, including a promoter (inducible or constitutive), enhancers, transcription or translation initiation sequence, transcription or translation termination sequence, secretion signals, Kozak sequence, cellular protein binding sequence, and the like.
  • BC-032 CTGCCCCGGGTTCCTCATTCTCZEGTTGGGTGCCTGCTGTACGGCCAAGGCET 47
  • BC-036 CTGCCCCGGGTTCCTCATTCTCZETTCTCACATCTGCTGTACGGCCAAGGCET 51
  • BC-041 CTGCCCCGGGTTCCTCATTCTCZZGTGGGATTGCTGCTGTACGGCCAAGGCET 56
  • BC-042 CTGCCCCGGGTTCCTCATTCTCZEAATGTACTACTGCTGTACGGCCAAGGCET 57
  • BC-044 CTGCCCCGGGTTCCTCATTCTCZFAGGATGATCCTGCTGTACGGCCAAGGCET 59
  • BC-045 CTGCCCCGGGTTCCTCATTCTCZETACTTGGCTCTGCTGTACGGCCAAGGCET 60
  • BC-046 CTGCCCCGGGTTCCTCATTCTCZEGTCGTCGAACTGCTGTACGGCCAAGGCET 61
  • BC-048 CTGCCCCGGGTTCCTCATTCTCZECCGTAAGTGCTGCTGTACGGCCAAGGCET 63
  • BC-049 CTGCCCCGGGTTCCTCATTCTCZFTGTCATAAGCTGCTGTACGGCCAAGGCET 64
  • BC-051 CTGCCCCGGGTTCCTCATTCTCZFAGCAGGAGTCTGCTGTACGGCCAAGGCET 66
  • BC-052 CTGCCCCGGGTTCCTCATTCTCZETAATTGTAACTGCTGTACGGCCAAGGCET 67
  • BC-056 CTGCCCCGGGTTCCTCATTCTCZECATGTCACCCTGCTGTACGGCCAAGGCET 71
  • BC-062 CTGCCCCGGGTTCCTCATTCTCZFGGTGAGGGTCTGCTGTACGGCCAAGGCET 77
  • BC-080 CTGCCCCGGGTTCCTCATTCTCZETTTACGGTGCTGCTGTACGGCCAAGGCET 95
  • BC-081 CTGCCCCGGGTTCCTCATTCTCZEAACGTCATTCTGCTGTACGGCCAAGGCET 96
  • BC-082 CTGCCCCGGGTTCCTCATTCTCZETGAAGGGAGCTGCTGTACGGCCAAGGCET 97
  • BC-084 CTGCCCCGGGTTCCTCATTCTCZECGGATGAACCTGCTGTACGGCCAAGGCET 99
  • BC-091 CTGCCCCGGGTTCCTCATTCTCZECGTATTGGGCTGCTGTACGGCCAAGGCET 106
  • BC-092 CTGCCCCGGGTTCCTCATTCTCZFAGGGATTACCTGCTGTACGGCCAAGGCET 107
  • a adapter (top 131 strand) GTCGGAGACACGCAGGGATGAGATGG*T*T
  • a adapter (top 140 strand) TTCCATCTCATCCCTGCGTGTCTCCGACTCAG
  • SEQ ID Blocker Oligo Sequences in 5' to 3' direction
  • Blocker BC 18 ATCGCAATTGCCTCTGAGTCGGAGACACGCAGGGATGAGATGG 161
  • Blocker BC 19 ATCGTCCGACTAACTGAGTCGGAGACACGCAGGGATGAGATGG 162
  • Blocker BC 20 ATCGATGGATCTGCTGAGTCGGAGACACGCAGGGATGAGATGG 163
  • Blocker BC 21 ATCGTAATTGCGACTGAGTCGGAGACACGCAGGGATGAGATGG 164
  • Blocker BC 22 ATCGCGTCTCGAACTGAGTCGGAGACACGCAGGGATGAGATGG 165
  • Blocker BC 23 ATCGTTCGTGGCACTGAGTCGGAGACACGCAGGGATGAGATGG 166
  • Blocker BC 24 ATCGAATGAGGTTCTGAGTCGGAGACACGCAGGGATGAGATGG 167
  • Blocker BC 25 ATCGTATCTCAGGCTGAGTCGGAGACACGCAGGGATGAGATGG 168
  • Blocker BC 26 ATCGAGGTTGTAACTGAGTCGGAGACACGCAGGGATGAGATGG 169
  • Blocker BC 27 ATCGCGGATGGTTCTGAGTCGGAGACACGCAGGGATGAGATGG 170
  • Blocker BC 28 ATCGATTCCGGATCTGAGTCGGAGACACGCAGGGATGAGATGG 171
  • Blocker BC 29 ATCGAGTGGTCGACTGAGTCGGAGACACGCAGGGATGAGATGG 172
  • Blocker BC 30 ATCGATAACCTCGCTGAGTCGGAGACACGCAGGGATGAGATGG 173
  • Blocker BC 31 ATCGCAGCTTGGACTGAGTCGGAGACACGCAGGGATGAGATGG 174
  • Blocker BC 32 ATCGTGTGTAAGACTGAGTCGGAGACACGCAGGGATGAGATGG 175
  • Blocker BC 33 ATCGTTCAATGAGAACTGAGTCGGAGACACGCAGGGATGAGATGG 176
  • Blocker BC 34 ATCGAACGATGCGACTGAGTCGGAGACACGCAGGGATGAGATGG 177
  • Blocker BC 35 ATCGACAATGGCTTACTGAGTCGGAGACACGCAGGGATGAGATGG 178
  • Blocker BC 36 ATCGACGATTCCTTCTGAGTCGGAGACACGCAGGGATGAGATGG 179
  • Blocker BC 37 ATCGACATTCTCAAGCTGAGTCGGAGACACGCAGGGATGAGATGG 180
  • Blocker BC 38 ATCGTCCGTCCTCCACTGAGTCGGAGACACGCAGGGATGAGATGG 181
  • Blocker BC 39 ATCGCCGATTGTTACTGAGTCGGAGACACGCAGGGATGAGATGG 182
  • Blocker BC 40 ATCGATTATGTCAGCTGAGTCGGAGACACGCAGGGATGAGATGG 183
  • Blocker BC 41 ATCGCGAAGTGGAACTGAGTCGGAGACACGCAGGGATGAGATGG 184
  • Blocker BC 42 ATCGATTCGTGCTCTGAGTCGGAGACACGCAGGGATGAGATGG 185
  • Blocker BC 43 ATCGCGGTGTCAAGCTGAGTCGGAGACACGCAGGGATGAGATGG 186
  • Blocker BC 44 ATCGCTGGCCTCCAACTGAGTCGGAGACACGCAGGGATGAGATGG 187
  • Blocker BC 45 ATCGAGGAAGCTCCACTGAGTCGGAGACACGCAGGGATGAGATGG 188
  • Blocker BC 46 ATCGTTCGGACTGACTGAGTCGGAGACACGCAGGGATGAGATGG 189
  • Blocker BC 47 ATCGTGGTTGCCTTACTGAGTCGGAGACACGCAGGGATGAGATGG 190
  • Blocker BC 48 ATCGTCTCTTAGAACTGAGTCGGAGACACGCAGGGATGAGATGG 191
  • Blocker BC 49 ATCGTTATGTTAGGACTGAGTCGGAGACACGCAGGGATGAGATGG 192
  • Blocker BC 50 ATCGCCATTGTCCGCTGAGTCGGAGACACGCAGGGATGAGATGG 193
  • Blocker BC 51 ATCGAATAGGCTCAACTGAGTCGGAGACACGCAGGGATGAGATGG 194
  • Blocker BC 52 ATCGTTCCATGCGGCTGAGTCGGAGACACGCAGGGATGAGATGG 195
  • Blocker BC 53 ATCGAGGATTGCCAGCTGAGTCGGAGACACGCAGGGATGAGATGG 196
  • Blocker BC 54 ATCGCGATTCTCCGGCTGAGTCGGAGACACGCAGGGATGAGATGG 197
  • Blocker BC 55 ATCGAGGAGGTGGACTGAGTCGGAGACACGCAGGGATGAGATGG 198
  • Blocker BC 56 ATCGAATTAATGCTGCTGAGTCGGAGACACGCAGGGATGAGATGG 199
  • Blocker BC 57 ATCGCCGTTGCCAGACTGAGTCGGAGACACGCAGGGATGAGATGG 200
  • Blocker BC 58 ATCGTGTTCTAGGACTGAGTCGGAGACACGCAGGGATGAGATGG 201
  • Blocker BC 59 ATCGAACATCAAGGACTGAGTCGGAGACACGCAGGGATGAGATGG 202
  • Blocker BC 60 ATCGAAGAGCTAGACTGAGTCGGAGACACGCAGGGATGAGATGG 203
  • Blocker BC 61 ATCGATCCGAGTGACTGAGTCGGAGACACGCAGGGATGAGATGG 204
  • Blocker BC 62 ATCGTGAAGCAGGAACTGAGTCGGAGACACGCAGGGATGAGATGG 205
  • Blocker BC 63 ATCGAACTCTAAGGCTGAGTCGGAGACACGCAGGGATGAGATGG 206
  • Blocker BC 64 ATCGTCGGAACTCAGCTGAGTCGGAGACACGCAGGGATGAGATGG 207
  • Blocker BC 65 ATCGATGTGCCAGGACTGAGTCGGAGACACGCAGGGATGAGATGG 208
  • Blocker BC 66 ATCGATGATTGCGGCTGAGTCGGAGACACGCAGGGATGAGATGG 209
  • Blocker BC 67 ATCGACTGGTAGGAACTGAGTCGGAGACACGCAGGGATGAGATGG 210
  • Blocker BC 68 ATCGAACTTCTTGACTGAGTCGGAGACACGCAGGGATGAGATGG 211
  • Blocker BC 69 ATCGCCAATTGAACTGAGTCGGAGACACGCAGGGATGAGATGG 212
  • Blocker BC 70 ATCGACCAGTAGGCTGAGTCGGAGACACGCAGGGATGAGATGG 213
  • Blocker BC 71 ATCGTCGGAGCCTCACTGAGTCGGAGACACGCAGGGATGAGATGG 214
  • Blocker BC 72 ATCGTGTGGCCTTCGCTGAGTCGGAGACACGCAGGGATGAGATGG 215
  • Blocker BC 73 ATCGACAGGCAGACTGAGTCGGAGACACGCAGGGATGAGATGG 216
  • Blocker BC 74 ATCGAACCGATCGCTGAGTCGGAGACACGCAGGGATGAGATGG 217
  • Blocker BC 75 ATCGTATTCCTGACTGAGTCGGAGACACGCAGGGATGAGATGG 218
  • Blocker BC 76 ATCGAGGTTCTTCCGCTGAGTCGGAGACACGCAGGGATGAGATGG 219
  • Blocker BC 77 ATCGAATCGCTTCGCTGAGTCGGAGACACGCAGGGATGAGATGG 220
  • Blocker BC 78 ATCGAGAATTGGCTGCTGAGTCGGAGACACGCAGGGATGAGATGG 221
  • Blocker BC 79 ATCGACAACCAGGCTGAGTCGGAGACACGCAGGGATGAGATGG 222
  • Blocker BC 80 ATCGCCTGCCTTCGACTGAGTCGGAGACACGCAGGGATGAGATGG 223
  • Blocker BC 81 ATCGCGAATGGCAGGCTGAGTCGGAGACACGCAGGGATGAGATGG 224
  • Blocker BC 82 ATCGAGATGCCAACTGAGTCGGAGACACGCAGGGATGAGATGG 225
  • Blocker BC 83 ATCGAATGTCCTAGCTGAGTCGGAGACACGCAGGGATGAGATGG 226
  • Blocker BC 84 ATCGTTATGGAAGCTGAGTCGGAGACACGCAGGGATGAGATGG 227
  • Blocker BC 85 ATCGTTGAGGCTGGCTGAGTCGGAGACACGCAGGGATGAGATGG 228
  • Blocker BC 86 ATCGAATAACCAAGCTGAGTCGGAGACACGCAGGGATGAGATGG 229
  • Blocker BC 87 ATCGTCCAGCCAACTGAGTCGGAGACACGCAGGGATGAGATGG 230
  • Blocker BC 88 ATCGAAGTGTTCGGCTGAGTCGGAGACACGCAGGGATGAGATGG 231
  • Blocker BC 89 ATCGAGATTCAGGACTGAGTCGGAGACACGCAGGGATGAGATGG 232
  • Blocker BC 90 ATCGCCGTGGTTAGCTGAGTCGGAGACACGCAGGGATGAGATGG 233
  • Blocker BC 91 ATCGCATCCTTCCGCTGAGTCGGAGACACGCAGGGATGAGATGG 234
  • Blocker BC 92 ATCGCGGTTCCTAGCTGAGTCGGAGACACGCAGGGATGAGATGG 235
  • Blocker BC 93 ATCGATTGGACAAGCTGAGTCGGAGACACGCAGGGATGAGATGG 236
  • Blocker BC 94 ATCGCT TGTCGGACTGAGTCGGAGACACGCAGGGATGAGATGG 23 7
  • Blocker BC 95 ATCGATCTGTCCGCTGAGTCGGAGACACGCAGGGATGAGATGG 23 8
  • Blocker BC 96 ATCGACCGCT TAACTGAGTCGGAGACACGCAGGGATGAGATGG 23 9
  • compositions and methods for capturing target polynucleotides where capture oligonucleotides can comprise an
  • capture oligonucleotides can be DNA, cDNA, RNA, or RNA/DNA hybrids. Capture oligonucleotides can be single- stranded or double- stranded nucleic acids or analogs thereof.
  • capture oligonucleotides can include nucleotide sequences that can hybridize to any portion of a target polynucleotide. In some embodiments, capture oligonucleotides can include nucleotide sequences that are fully or partially complementary to any portion of a target polynucleotide.
  • capture oligonucleotides can include sequences that are complementary to chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified (e.g., PCR amplified), cDNA, RNA such as precursor mRNA or mRNA, oligonucleotide, or any type of nucleic acid library.
  • capture oligonucleotides can include sequences that are complementary to any sequence from any organism such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, or viruses.
  • capture oligonucleotides can include sequences that are complementary to any sequence from normal or diseased cells or tissues or organs. In some embodiments, when multiple capture oligonucleotides hybridize to a target polynucleotide, they can hybridize to regions that overlap or are not overlapping.
  • capture oligonucleotides can include nucleotide sequences that are fully complementary (e.g., base pairing A-T and/or C/G) or partially complementary (e.g., mis-match pairing A/C or G, T/C or G, C/A or T, or G/A or T) to any portion of the
  • capture oligonucleotides can include degenerate sequences. In some embodiments, capture oligonucleotides can include one or more inosine residues.
  • Capture oligonucleotides can be any length, including about 5-25 bases, or about 25-50 bases, or about 50-75 bases, or about 75-100 bases, or about 100-125 bases, or about 125-150 bases, or about 150-175 bases, or about 175-200 bases, or about 200-225 bases, or about 225-250 bases, or about 250-275 bases, or about 275-300 bases, or about 300-500 bases, or longer.
  • polynucleotides can be hybridized with about 500,000 - 1 million different capture oligonucleotides, or with about 1-1.5 million different capture oligonucleotides, or with about 1.5-2 million different capture oligonucleotides, or with about 2- 2.5 million different capture oligonucleotides, or with about 2.5 - 3 million different capture oligonucleotides, or more.
  • capture oligonucleotides can include at least one scissile linkage.
  • a scissile linkage can be susceptible to cleavage or degradation by an enzyme or chemical compound.
  • capture oligonucleotides can include at least one phosphorothiolate, phosphorothioate, and/or phosphoramidate linkage.
  • the present teachings provide compositions and methods for capturing target polynucleotides, where capture oligonucleotides can include one member of a binding partner.
  • molecules that function as binding partners include: biotin (and its derivatives) and their binding partner avidin moieties, streptavidin moieties (and their derivatives); His-tags which bind with nickel, cobalt or copper; cysteine, histidine, or histidine patch which bind Ni-NTA; maltose which binds with maltose binding protein (MBP); lectin-carbohydrate binding partners; calcium-calcium binding protein (CBP); acetylcholine and receptor- acetylcholine; protein A and binding partner anti-FLAG antibody; GST and binding partner glutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor) protein; antigen or epitope tags which bind to antibody or antibody fragments
  • An avidin moiety can include an avidin protein, as well as any derivatives, analogs and other non-native forms of avidin that can bind to biotin moieties.
  • Other forms of avidin moieties include native and recombinant avidin and streptavidin as well as derivatized molecules, e.g. nonglycosylated avidins, N-acyl avidins and truncated streptavidins.
  • avidin moiety includes deglycosylated forms of avidin, bacterial streptavidins produced by Streptomyces (e.g., Streptomyces avidinii), truncated streptavidins, recombinant avidin and streptavidin as well as to derivatives of native, deglycosylated and recombinant avidin and of native, recombinant and truncated streptavidin, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin, and the commercial products ExtrAvidinTM, CaptavidinTM, NeutravidinTM and Neutralite
  • the present teachings provide compositions and methods for capturing target polynucleotides, where capture oligonucleotides that are attached to a member of a binding partner (e.g., biotin) can bind another member of a binding partner (e.g., avidin-like, such as streptavidin) which is attached to a surface.
  • a surface can be an outer or top-most layer or boundary of an object.
  • a surface can be interior to the boundary of an object.
  • a surface can be porous or non-porous.
  • a surface can be a planar surface, as well as concave, convex, or any combination thereof.
  • a surface can be a bead, particle, microparticle, sphere, filter, flowcell, or gel.
  • a surface includes the inner walls of a capillary, a channel, a well, groove, channel, reservoir.
  • a surface can include texture (e.g., etched, cavitated, pores, three-dimensional scaffolds or bumps).
  • a surface can be made from materials such as glass, borosilicate glass, silica, quartz, fused quartz, mica, polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics, silicon, semiconductor, high refractive index dielectrics, crystals, gels, polymers, or films (e.g., films of gold, silver, aluminum, or diamond).
  • a surface can be magnetic or paramagnetic (e.g., magnetic or paramagnetic microparticles).
  • paramagnetic microparticles can be paramagnetic beads attached with streptavidin (e.g., DynabeadsTM M-270 from Invitrogen, Carlsbad, CA). Additional Reactions
  • the present teachings provide compositions and methods for capturing target polynucleotides, where capture duplexes, enriched target polynucleotides and/or released target polynucleotides can be subjected to further manipulations.
  • further manipulations can include nucleic acid manipulations. Nucleic acid manipulation can be conducted in any combination and in any order and include: chemical modification, size- selection, end repairing, tailing, adaptor-joining, ligation, nick repairing, purification, nick translation, amplification, surface attachment and/or sequencing. In some embodiments, any of these nucleic acid manipulations can be omitted or can be repeated.
  • reduced complexity target polynucleotides can be modified to attach to a surface.
  • reduced complexity target polynucleotides can be amino- modified for attachment to a surface (e.g., particles or a planar surface).
  • an amino-modified nucleic acid can be attached to a surface that is coated with a carboxylic acid.
  • an amino-modified nucleic acid can be reacted with EDC (or ED AC) for attachment to a carboxylic acid coated surface (with or without NHS).
  • target polynucleotides can be attached to particles, such as Ion SphereTM particles (Life
  • reduced complexity target polynucleotides can be amplified.
  • amplification can be conducted using at least one amplification primer that can hybridize to either strand or any portion of the polynucleotide constructs, including a nucleic acid adaptor or a target polynucleotide.
  • amplification can include thermo-cycling amplification or isothermal amplification reactions.
  • amplification can be conducted with polymerase that are thermo-stable or thermo-labile.
  • amplification can be conducted as a PCR reaction.
  • reduced complexity target polynucleotides can be subjected to any size-selection procedure to obtain any desired size range. In some embodiments, reduced complexity target polynucleotides are not size- selected.
  • nucleic acid size selection method includes without limitation: solid phase adherence or immobilization; electrophoresis, such as gel electrophoresis; and chromatography, such as HPLC and size exclusion chromatography.
  • a solid phase adherence/immobilization methods involves paramagnetic beads coated with a chemical functional group that interacts with nucleic acids under certain ionic strength conditions with or without polyethylene glycol or polyalkylene glycol.
  • solid phase adherence/immobilization methods include but are not limited to: SPRI (Solid Phase Reversible Immobilization) beads from Agencourt (see Hawkins 1995 Nucleic Acids Research 23:22) which are carboxylate-modified paramagnetic beads; MAGNA PURE magnetic glass particles (Roche Diagnostics, Hoffmann-La Roche Ltd.); MAGNESIL magnetic bead kit from Promega; BILATEST magnetic bead kit from Bilatec AG;
  • MAGTRATION paramagnetic system from Precision System Science, Inc.; MAG BIND from Omega Bio-Tek; MAGPREP silica from Merck/Estapor; SNARe DNA purification system from Bangs; CHEMAGEN M-PVA beads from CHEMAGEN; and magnetic beads from Aline Bioscience (DNA Purification Kit).
  • size-selected nucleic acids can be about 50-250 bp, or about 250-500 bp, or about 500-750 bp, or about 750-1000 bp, or about 1-5 kb, or about 5-10 kb, or about 10-25 kb, or about 25-50 kb, or about 50-60 kb or longer.
  • reduced complexity target polynucleotides may be desirable.
  • reduced complexity target polynucleotides can have a first end, a second end, or an internal portion, having undesirable features, such as nicks, overhang ends, ends lacking a phosphorylated end, ends having a phosphorylated end, or nucleic acid fragments having apurinic or apyrimidinic residues.
  • enzymatic reactions can be conducted to repair one or more ends or internal portions.
  • reduced complexity target polynucleotides can be subjected to enzymatic reactions to convert overhang ends to blunt ends, or to phosphorylate or de-phosphorylate the 5' end of a strand, or to close nicks, to repair oxidized purines or pyrimidines, to repair deaminated cytosines, or to hydrolyze the apurinic or apyrimidinic residues.
  • repairing or end-repairing target polynucleotides includes contacting nucleic acid fragments with: an enzyme to close single- stranded nicks in duplex DNA (e.g., T4 DNA ligase); an enzyme to phosphorylate the 5' end of at least one strand of a duplex DNA (e.g., T4 polynucleotide kinase); an enzyme to remove a 5' or 3'phosphate (e.g., any phosphatase enzyme, such as calf intestinal alkaline phosphatase, bacterial alkaline phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase, and placental alkaline phosphatase); an enzyme to remove 3' overhang ends (e.g., DNA polymerase I, Large (Klenow) fragment, T4 DNA polymerase, mung bean nuclease); an enzyme to fill-in 5' overhang ends (e.g., T4 DNA polymerase I,
  • a repairing or end-repairing reaction can be supplemented with additional repairing enzymes in any combination and in any amount, including: endonuclease IV (apurinic- apyrimidinic removal), Bst DNA polymerase (5'>3' exonuclease for nick translation), formamidopyrimidine DNA glycosylase (FPG) (e.g., base excision repair for oxidize purines), uracil DNA glycosylase (uracil removal), T4 endonuclease V (pyrimidine removal) and/or endonuclease VIII (removes oxidized pyrimidines).
  • FPG formamidopyrimidine DNA glycosylase
  • a repairing or end- repairing reaction can be conducted in the presence of appropriate co-factors, including dNTPs, NAD, (NH 4 ) 2 S0 4 , KC1, and/or MgS0 4 .
  • reduced complexity target polynucleotides can be subjected to any purification procedure to remove non-desirable materials (buffers, salts, enzymes, primer- dimers, or excess adaptors or primers).
  • a purification procedure can be conducted between any two steps to remove buffers, salts, enzymes, adaptors, non-reacted nucleic acid fragments, and the like. Purification procedures include without limitation: bead purification, column purification, gel electrophoresis, dialysis, alcohol precipitation, and size- selective PEG precipitation.
  • reduced complexity target polynucleotides can be subjected to a tailing reaction (e.g., non-template-dependent terminal transferase reaction).
  • a non-template-dependent terminal transferase reaction can be catalyzed by a Taq polymerase, Tfi DNA polymerase, 3' exonuclease minus- large (Klenow) fragment, or 3' exonuclease minus- T4 polymerase.
  • reduced complexity target polynucleotides can be subject to a nick repairing or nick repair reaction.
  • a nick repair reaction can be catalyzed by a nick repair polymerase such as Taq DNA polymerase, Bst DNA polymerase, Platinum® Pfx DNA polymerase (Invitrogen), Tfi Exo(-) DNA polymerase (Invitrogen) or Phusion® Hot Start High-Fidelity DNA polymerase (New England Biolabs).
  • the nick repair enzyme can be used to extend the nucleic acid strand from the site of the nick to the original termini of the adaptor sequence.
  • nucleotides (or analogs thereof) used for any nucleic acid manipulation can be attached to a label.
  • a label comprises a detectable moiety.
  • a label can generate, or cause to generate, a detectable signal.
  • a detectable signal can be generated from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events).
  • a proximity event can include two reporter moieties approaching each other, or associating with each other, or binding each other.
  • a detectable signal can be detected optically, electrically, chemically, enzymatically, thermally, or via mass spectroscopy or Raman spectroscopy.
  • a label can include compounds that are luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent or electrochemical.
  • a label can include compounds that are fluorophores, chromophores, radioisotopes, haptens, affinity tags, atoms or enzymes.
  • the label comprises a moiety not typically present in naturally occurring nucleotides.
  • the label can include fluorescent, luminescent or radioactive moieties.
  • reduced complexity target polynucleotides can be sequenced by any sequencing method, including sequencing-by-synthesis, ion-based sequencing involving the detection of sequencing byproducts using field effect transistors (e.g., FETs and ISFETs), chemical degradation sequencing, ligation-based sequencing, hybridization sequencing, pyrophosphate detection sequencing, capillary electrophoresis, gel electrophoresis, next- generation, massively parallel sequencing platforms, sequencing platforms that detect hydrogen ions or other sequencing by-products, and single molecule sequencing platforms.
  • a sequencing reaction can be conducted using at least one sequencing primer that can hybridize to any portion of the polynucleotide constructs, including a nucleic acid adaptor or a target polynucleotide.
  • reduced complexity target polynucleotides produced by the methods described herein can be used in any nucleic acid sequencing workflow, including sequencing by oligonucleotide probe ligation and detection (e.g., SOLiDTM from Life
  • probe-anchor ligation sequencing e.g., Complete
  • GenomicsTM or PolonatorTM sequencing- by-synthesis
  • sequencing- by-synthesis e.g., Genetic Analyzer and HiSeqTM, from niumina
  • pyrophosphate sequencing e.g., Genome Sequencer FLX from 454 Life Sciences
  • ion-sensitive sequencing e.g., Personal Genome Machine (PGMTM) and Ion ProtonTM Sequencer, both from Ion Torrent Systems, Inc.
  • single molecule sequencing platforms e.g., HeliScopeTM from HelicosTM.
  • genomic DNA can be isolated from a cell, tissue or organ.
  • genomic DNA can be fragmented via enzymatic, chemical or physical fragmentation methods.
  • fragmented DNA e.g., polynucleotides
  • a collection of non-target and target polynucleotide constructs can form a nucleic acid library.
  • a nucleic acid library can be amplified.
  • a nucleic acid library can be denatured to form a single- stranded library.
  • a single stranded nucleic acid library can be hybridized to at least one blocker oligonucleotide and at least one biotinylated capture oligonucleotide and non-specific oligonucleotides (e.g., human Cot-1 DNA), under suitable hybridization conditions to form capture duplexes having target polynucleotides hybridized to capture oligonucleotides.
  • suitable hybridization conditions can include about 40-50 °C for about 60-75 hours.
  • paramagnetic streptavidin beads can be reacted with the capture duplexes to recover the enriched target polynucleotides.
  • the paramagnetic streptavidin beads and capture duplexes can be reacted at about 40-50 °C for about 15-75 minutes to form a bead- duplex complex.
  • the bead-duplex complex can be washed with a buffer (e.g., high stringency wash buffer) to remove un-hybridized nucleic acids to enrich for target polynucleotides hybridized to biotinylated capture oligonucleotides.
  • the enriched target polynucleotides can be denatured to release single-stranded target
  • enriched target polynucleotides can be amplified.
  • amplified target polynucleotides can be conjugated to microparticles and amplified to form microparticles templated with clonal copies of the target polynucleotide.
  • target polynucleotides attached to the microparticles can be sequenced in any sequencing platform (e.g., Ion Torrent PGMTM or ProtonTM sequencer (Ion TorrentTM Systems, Life Technologies Corporation).
  • one or more reduced complexity target polynucleotides produced according to the present teachings can be sequenced using methods that detect one or more byproducts of nucleotide incorporation.
  • the detection of polymerase extension by detecting physicochemical byproducts of the extension reaction can include pyrophosphate, hydrogen ion, charge transfer, heat, and the like, as disclosed, for example, in Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006); Purushothaman et al., IEEE ISCAS, IV-169-172; Rothberg et al, U.S. Patent Publication No. 2009/0026082; Anderson et al, Sensors and
  • reactions involving the generation and detection of ions are widely performed.
  • the use of direct ion detection methods to monitor the progress of such reactions can simplify many current biological assays.
  • template-dependent nucleic acid synthesis by a polymerase can be monitored by detecting hydrogen ions that are generated as natural byproducts of nucleotide incorporations catalyzed by the polymerase.
  • Ion-sensitive sequencing also referred to as "pH-based” or "ion-based” nucleic acid sequencing
  • the nucleic acid to be sequenced can be captured in a microwell, and nucleotides can be flowed across the well, one at a time, under nucleotide incorporation conditions.
  • the polymerase incorporates the appropriate nucleotide into the growing strand, and the hydrogen ion that is released can change the pH in the solution, which can be detected by an ion sensor that is coupled with the well.
  • This technique does not require labeling of the nucleotides or expensive optical components, and allows for far more rapid completion of sequencing runs. Examples of such ion-based nucleic acid sequencing methods and platforms include the Ion Torrent PGMTM or ProtonTM sequencer (Ion TorrentTM Systems, Life Technologies Corporation).
  • target polynucleotides produced using the methods, systems and kits of the present teachings can be used as a substrate for a biological or chemical reaction that is detected and/or monitored by a sensor including a field-effect transistor (FET).
  • FET field-effect transistor
  • the FET is a chemFET or an ISFET.
  • a "chemFET” or chemical field-effect transistor is a type of field effect transistor that acts as a chemical sensor. It is the structural analog of a MOSFET transistor, where the charge on the gate electrode is applied by a chemical process.
  • An "ISFET” or ion-sensitive field-effect transistor is used for measuring ion
  • the FET may be a FET array.
  • an "array" is a planar arrangement of elements such as sensors or wells.
  • the array may be one or two dimensional.
  • a one dimensional array can be an array having one column (or row) of elements in the first dimension and a plurality of columns (or rows) in the second dimension. The number of columns (or rows) in the first and second dimensions may or may not be the same.
  • the FET or array can comprise 102, 103, 104, 105, 106, 107 or more FETs.
  • one or more microfluidic structures can be fabricated above the FET sensor array to provide for containment and/or confinement of a biological or chemical reaction.
  • the microfluidic structure(s) can be configured as one or more wells (or microwells, or reaction chambers, or reaction wells, as the terms are used interchangeably herein) disposed above one or more sensors of the array, such that the one or more sensors over which a given well is disposed detect and measure analyte presence, level, and/or concentration in the given well.
  • Microwells or reaction chambers are typically hollows or wells having well-defined shapes and volumes which can be manufactured into a substrate and can be fabricated using conventional microfabrication techniques, e.g. as disclosed in the following references: Doering and Nishi, Editors, Handbook of Semiconductor Manufacturing Technology, Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMS and Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al, Silicon Micromachining (Cambridge University Press, 2004); and the like. Examples of configurations (e.g. spacing, shape and volumes) of microwells or reaction chambers are disclosed in Rothberg et al, U.S. patent publication 2009/0127589; Rothberg et al, U.K. patent application GB24611127.
  • the biological or chemical reaction can be performed in a solution or a reaction chamber that is in contact with or capacitively coupled to a FET such as a chemFET or an ISFET.
  • a FET such as a chemFET or an ISFET.
  • the FET (or chemFET or ISFET) and/or reaction chamber can be an array of FETs or reaction chambers, respectively.
  • a biological or chemical reaction can be carried out in a two- dimensional array of reaction chambers, wherein each reaction chamber can be coupled to a FET, and each reaction chamber is no greater than 10 ⁇ (i.e., 1 pL) in volume. In some embodiments each reaction chamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pL in volume.
  • a reaction chamber can optionally be 22, 32, 42, 52, 62, 72, 82, 92, or 102 square microns in cross-sectional area at the top.
  • the array has at least 102, 103, 104, 105, 106, 107,108, 109, or more reaction chambers. In some embodiments, the reaction chambers can be capacitively coupled to the FETs.
  • FET arrays as used in various embodiments according to the disclosure can be fabricated according to conventional CMOS fabrications techniques, as well as modified CMOS fabrication techniques and other semiconductor fabrication techniques beyond those
  • CMOS fabrication conventionally employed in CMOS fabrication. Additionally, various lithography techniques can be employed as part of an array fabrication process.
  • Exemplary FET arrays suitable for use in the disclosed methods, as well as microwells and attendant fluidics, and methods for manufacturing them, are disclosed, for example, in U.S. Patent Publication No. 20100301398; U.S. Patent Publication No. 20100300895; U.S. Patent Publication No. 20100300559; U.S. Patent Publication No. 20100197507, U.S. Patent
  • the disclosed methods, compositions, systems, apparatuses and kits can be used for carrying out label-free nucleic acid sequencing, and in particular, ion-based nucleic acid sequencing.
  • label-free detection of nucleotide incorporation has been described in the literature, including the following references that are incorporated by reference: Rothberg et al, U.S. patent publication 2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86 (2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006).
  • nucleotide incorporations are determined by measuring natural byproducts of polymerase-catalyzed extension reactions, including hydrogen ions, polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase).
  • ion-based nucleic acid sequencing methods and platforms include the Ion Torrent PGMTM or ProtonTM sequencer (Ion TorrentTM Systems, Life Technologies Corporation).
  • the disclosure relates generally to methods for sequencing the reduced complexity target polynucleotides produced by the teachings provided herein.
  • the disclosure relates generally to a method for obtaining sequence information from reduced complexity target polynucleotides, comprising: (a) conducting reactions to obtain reduced complexity target polynucleotides; and (b) performing template- dependent nucleic acid synthesis using at least one of the reduced complexity target polynucleotides produced during step (a) as a template.
  • capturing for target polynucleotides can include hybridizing a plurality of polynucleotide to one or more blocker oligonucleotides and with one or more capture oligonucleotides to form duplexes having a target polynucleotide hybridized to a capture oligonucleotide.
  • the methods can further comprise separating the duplexes from nucleic acids in the sample that are not part of a duplex to obtain enriched target polynucleotides.
  • the template-dependent synthesis includes incorporating one or more nucleotides in a template-dependent fashion into a newly synthesized nucleic acid strand.
  • the methods can further include producing one or more ionic byproducts of such nucleotide incorporation.
  • the methods can further include detecting the incorporation of the one or more nucleotides into the sequencing primer.
  • the detecting can include detecting the release of hydrogen ions.
  • the disclosure relates generally to a method for sequencing a nucleic acid, comprising: (a) producing a plurality of reduced complexity target polynucleotides according to the methods disclosed herein; (b) disposing a plurality of reduced complexity target polynucleotides into a plurality of reaction chambers, wherein one or more of the reaction chambers are in contact with a field effect transistor (FET).
  • FET field effect transistor
  • the method further includes contacting at least one of the reduced complexity target polynucleotides disposed into one of the reaction chambers with a polymerase, thereby synthesizing a new nucleic acid strand by sequentially incorporating one or more nucleotides into a nucleic acid molecule.
  • the method further includes generating one or more hydrogen ions as a byproduct of such nucleotide incorporation.
  • the method further includes detecting the incorporation of the one or more nucleotides by detecting the generation of the one or more hydrogen ions using the FET.
  • the detecting includes detecting a change in voltage and/or current at the at least one FET within the array in response to the generation of the one or more hydrogen ions.
  • the FET can be selected from the group consisting of: ion- sensitive FET (isFET) and chemically- sensitive FET (chemFET).
  • One exemplary system involving sequencing via detection of ionic byproducts of nucleotide incorporation is the Ion Torrent PGMTM or ProtonTM sequencer (Life Technologies), which is an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions produced as a byproduct of nucleotide incorporation. Typically, hydrogen ions are released as byproducts of nucleotide incorporations occurring during template-dependent nucleic acid synthesis by a polymerase.
  • the Ion Torrent PGMTM or ProtonTM sequencer detects the nucleotide incorporations by detecting the hydrogen ion byproducts of the nucleotide incorporations.
  • the Ion Torrent PGMTM or ProtonTM sequencer can include a plurality of nucleic acid templates to be sequenced, each template disposed within a respective sequencing reaction well in an array.
  • the wells of the array can each be coupled to at least one ion sensor that can detect the release of H + ions or changes in solution pH produced as a byproduct of nucleotide incorporation.
  • the ion sensor comprises a field effect transistor (FET) coupled to an ion- sensitive detection layer that can sense the presence of H + ions or changes in solution pH.
  • FET field effect transistor
  • the ion sensor can provide output signals indicative of nucleotide incorporation which can be represented as voltage changes whose magnitude correlates with the H + ion concentration in a respective well or reaction chamber.
  • nucleotide types can be flowed serially into the reaction chamber, and can be incorporated by the polymerase into an extending primer (or polymerization site) in an order determined by the sequence of the template.
  • Each nucleotide incorporation can be accompanied by the release of H + ions in the reaction well, along with a concomitant change in the localized pH.
  • the release of H + ions can be registered by the FET of the sensor, which produces signals indicating the occurrence of the nucleotide incorporation. Nucleotides that are not incorporated during a particular nucleotide flow may not produce signals.
  • the amplitude of the signals from the FET can also be correlated with the number of nucleotides of a particular type incorporated into the extending nucleic acid molecule thereby permitting homopolymer regions to be resolved.
  • multiple nucleotide flows into the reaction chamber along with incorporation monitoring across a multiplicity of wells or reaction chambers can permit the instrument to resolve the sequence of many nucleic acid templates simultaneously.
  • Further details regarding the compositions, design and operation of the Ion Torrent PGMTM or ProtonTM sequencer can be found, for example, in U.S. Patent Application Ser. No. 12/002781, now published as U.S. Patent Publication No. 2009/0026082; U.S. Patent Application Ser. No.
  • nucleotide In a typical embodiment of ion-based nucleic acid sequencing, nucleotide
  • templates each having a primer and polymerase operably bound can be loaded into reaction chambers (such as the microwells disclosed in Rothberg et al, cited herein), after which repeated cycles of nucleotide addition and washing can be carried out.
  • such templates can be attached as clonal populations to a solid support, such as particles, bead, or the like, and said clonal populations are loaded into reaction chambers.
  • operably bound means that a primer is annealed to a template so that the primer's 3' end may be extended by a polymerase and that a polymerase is bound to such primer-template duplex, or in close proximity thereof so that binding and/or extension takes place whenever nucleotides are added.
  • the polymerase can extend the primer by
  • incorporating added nucleotide only if the next base in the template is the complement of the added nucleotide. If there is one complementary base, there is one incorporation, if two, there are two incorporations, if three, there are three incorporations, and so on. With each such
  • the production of hydrogen ions is monotonically related to the number of contiguous complementary bases in the template (as well as the total number of template molecules with primer and polymerase that participate in an extension reaction).
  • the number of hydrogen ions generated, and therefore the magnitude of the local pH change can be proportional to the number of contiguous identical complementary bases. If the next base in the template is not complementary to the added nucleotide, then no incorporation occurs and no hydrogen ion is released.
  • an additional step can be performed, in which an unbuffered wash solution at a predetermined pH is used to remove the nucleotide of the previous step in order to prevent misincorporations in later cycles.
  • an additional step can be performed wherein the reaction chambers are treated with a nucleotide-destroying agent, such as apyrase, to eliminate any residual nucleotides remaining in the chamber, which may result in spurious extensions in subsequent cycles.
  • nucleotides are added sequentially to the reaction chambers, so that each reaction can be exposed to the different nucleotides one at a time.
  • nucleotides can be added in the following sequence: dATP, dCTP, dGTP, dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposure followed by a wash step.
  • the cycles may be repeated for 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 750 times, or more, depending on the length of sequence information desired.
  • sequencing can be performed according to the user protocols supplied with the PGMTM or ProtonTM sequencer.
  • Example 3 provides one exemplary protocol for ion-based sequencing using the Ion Torrent PGMTM sequencer (Ion TorrentTM Systems, Life Technologies, CA).
  • the present teachings provide systems for capturing target polynucleotides, comprising any combination of: blocker oligonucleotides, capture
  • oligonucleotides conjugated or not to a binding moiety
  • first nucleic acid adaptors conjugated or not to a binding moiety
  • nucleic acid second adaptors conjugated or not to a binding partner moiety
  • beads conjugated or not to a binding partner moiety
  • hybridization solutions and/or washing solutions.
  • a system can include all or some of these components.
  • systems for generating reduced complexity target polynucleotides can further comprise any combination of: buffers; cations; size- selection reagents; one or more end-repairing enzyme(s); one or more repairing enzyme(s); one or more nick repair enzymes; one or more ligation enzyme(s); reagents for nucleic acid purification; reagents for nucleic acid amplification; endonuclease(s); polymerase(s); kinase(s); phosphatase(s); and/or nuclease(s).
  • kits for capturing target polynucleotides include any reagent that can be used to capture target polynucleotides from a nucleic acid sample.
  • kits include any combination of: blocker oligonucleotides, capture oligonucleotides (conjugated or not to a binding moiety), first nucleic acid adaptors, second nucleic acid adaptors, beads (conjugated or not to a binding partner moiety), hybridization solutions, and/or washing solutions.
  • a kit can include all or some of these components.
  • a kit for generating reduced complexity target polynucleotides can further comprise any combination of: buffers; cations; size-selection reagents; one or more end-repairing enzyme(s); one or more repairing enzyme(s); one or more nick repair enzymes; one or more ligation enzyme(s); reagents for nucleic acid purification; reagents for nucleic acid amplification; endonuclease(s); polymerase(s); kinase(s); phosphatase(s); and/or nuclease(s).

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Abstract

Selon certains modes de réalisation, la présente invention concerne des compositions, des systèmes, des procédés et des nécessaires pour la réduction de la complexité de séquences nucléotidiques dans un échantillon d'acide nucléique, lesdits procédés comportant les étapes suivantes : l'hybridation d'une pluralité de constructions polynucléotidiques à au moins un oligonucléotide bloqueur et à au moins un oligonucléotide de capture, la pluralité de constructions polynucléotidiques comprenant une pluralité de polynucléotides dont chaque polynucléotide est joint à au moins un adaptateur d'acide nucléique, le ou les adaptateurs d'acide nucléique pouvant s'hybrider au ou aux oligonicléotides bloqueurs, et le ou les oligonucléotides de capture pouvant s'hybrider à au moins une partie de polynucléotides cibles qui sont une sous-population de la pluralité de polynucléotides, afin de produire un duplex de capture.
PCT/US2012/046624 2011-07-14 2012-07-13 Réduction de la complexité d'acide nucléique WO2013010062A2 (fr)

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