(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`OQ UTA ATTAAAAA
`
`(43) International Publication Date
`9 January 2014 (09.01.2014)
`
`WIPO!IPCT
`
`\=
`
`(10) International Publication Number
`WO 2014/008447 Al
`
`GD)
`
`International Patent Classification:
`C12N 15/10 (2006.01)
`C12@ 1/68 (2006.01)
`CI2N 15/11 (2006.01)
`C07H 21/04 (2006.01)
`
`QD
`
`International Application Number:
`
`PCT/US2013/049402
`
`(22)
`
`International Filing Date:
`
`3 July 2013 (03.07.2013)
`
`(25)
`
`(26)
`
`(30)
`
`(7)
`
`(72)
`
`Filing Language:
`
`Publication Language:
`
`Priority Data:
`61/667,919
`61/745,435
`
`3 July 2012 (03.07.2012)
`21 December 2012 (21.12.2012)
`
`English
`
`English
`
`US
`US
`
`INTEGRATED DNA TECHNOLOGIES,
`Applicants:
`INC.
`[US/US]; 1710 Commercial Park, Coralville,
`IA
`52241
`(US),
`FOUNDATION MEDICINE,
`INC.
`[US/US]; One Kendall Square, Suite B3501, Cambridge,
`MA 02139 (US).
`
`Inventors: BEHLKE, Mark, Aaron; 961 Timber Ridge
`Court, Coralville, TA 52241
`(US). HAVENS, John,
`Robert; 8749 William Cody Drive, Evergreen, CO 80439
`(US). ROSE, Scott Daniel; 1180 Mesquite Drive, Cor-
`
`(74)
`
`(81)
`
`alville, [A 52241 (US). JAROSZ, Mirna; 962 Clark Way,
`Palo Alto, CA 94304 (US). ZWIRKO, Zachary; 11 Com-
`monwealth Court, Apartment #5, Brighton, MA 02135
`(US). LIPSON, Doron; 142 Middlesex Road, #1, Chestnut
`Hill, MA 02457 (US). JUHN, Frank, Soo; 85 Beals Street,
`Brookline, MA 02446 (US).
`
`Agents: CELANDER, Daniel, W. et al.; Klintworth &
`Rozenblat IP LLC, 850 West Jackson Boulevard, Suite
`525, Chicago, IL 60607 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM,GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM,PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`
`(54) Title: TM-ENHANCED BLOCKING OLIGONUCLEOTIDES AND BAITS FOR IMPROVED TARGET ENRICHMENT
`AND REDUCED OFF-TARGET SELECTION
`
`[Continued on next page]
`
`(57) Abstract: The invention is directed to modified oligonucleotide com-
`positions and methods for selectively reducing unwanted nucleic acid con-
`taminants and enriching for desired nucleic acid targets from complex gen-
`omic nucleic acid mixtures for sequencing applications. The modified oligo-
`nucleotide compositions include one or more modified groups that increase
`the Tm of the resultant oligonucleotide composition.
`
`GENOMIC DNA
`
`
`
`~~A09
`
`a v
`
`SHEARING,
`ADAPTERLIGATION,
`WALAL}
`
`POR (OPT!
`
`
`
`
`
`ce
`7
`12
`
`109--—EZEZa
`Oe 103 (DOUBLE-STRANDED)
`
`2 POND
`SOLUTION RYBRIDIZATION
`
`we (SINGLE-STRANDED)
`
`
`
`CATCH
`
`
`
`eas 107 (DES/RED TARGETS)
`
`
`
`
`
`db SEQUENCING
`FIG. 7
`
`
`
`
`
`wo2014/008447A1IMINILTINAITAINNIAITUNANATA
`
`

`

`WO 2014/008447 A1IfMIIMNTHIIT INTEIMTMTT TATA TAT MTAA
`
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, Published:
`Ehneeeoe eRtyeT a ty —__with international search report (Art. 21(3))
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SL, SK, —__with sequencelisting part ofdescription (Rule 5.2(a))
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, KM, ML, MR, NE, SN, TD, TG).
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`Tm-ENHANCED BLOCKING OLIGONUCLEOTIDES AND BAITS FOR IMPROVED
`TARGET ENRICHMENT AND REDUCED OFF-TARGET SELECTION
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[01]
`
`This application claims benefit of priority under 35 U.S.C. 119 from U.S. Provisional
`
`Application No. 61/667,919, filed July 3, 2012 and entitled “METHODS AND
`
`COMPOSITIONS FOR REDUCING OFF-TARGET SELECTION” and U.S. Provisional
`
`Application No. 61/745,435, filed December 21, 2012 and entitled “TmM-ENHANCED
`
`BLOCKING OLIGONUCLEOTIDES AND BAITS FOR IMPROVED TARGET
`
`ENRICHMENT IN MASSIVELY PARALLEL SEQUENCING EXPERIMENTS,”the
`
`contents of both which are incorporated by reference herein in their entireties.
`
`FIELD OF THE INVENTION
`
`[02]
`
`This invention relates to modified oligonucleotide compositions and their use in
`
`methods for nucleic acid selection and sequencing. In particular, the invention pertains to
`
`Tm-enhanced oligonucleotides as blockers and baits, as well as other reagents for improved
`
`target enrichment and reduced off-target selection. The oligonucleotide compositions and
`
`reagents find robust applications for preparing nucleic acid templates for next generation
`
`sequencing applications.
`
`BACKGROUND OF THE INVENTION
`
`[03] Nucleic acid hybridization has a significant role in biotechnology applications
`
`pertaining to identification, selection, and sequencing of nucleic acids. Sequencing
`
`applications with genomic nucleic acids as the target materials demand oneto select nucleic
`
`acid targets of interest from a highly complex mixture. The quality of the sequencing efforts
`
`depends onthe efficiency of the selection process, which, in turn, relies upon how well
`
`nucleic acid targets can be enriched relative to non-target sequences.
`
`[04]
`
`A varicty of mcthods have been usedto enrich for desired sequences from a complex
`
`pool of nucleic acids, such as genomic DNA or cDNA. These methods include the
`
`polymerase chain reaction (PCR), molecular inversion probes (MIPs), or sequence capture by
`
`hybrid formation (“hybrid capture;” See, for example, Mamanova,L., Coffey, A.J., Scott,
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`C.E., Kozarewa, I., Turner, E.H., Kumar, A., Howard, E., Shendure, J. and Turner, D.J.
`
`(2010) “Target-cnrichment strategics for ncxt-gcencration sequencing,” Nat. Methods 7:111-
`
`118.). Hybrid capture offers advantages over other methodsin that this method requires
`
`fewer enzymatic amplification or manipulation procedures of the target nucleic acid as
`
`compared to the other methods. The hybrid capture method introduces fewer errors into the
`
`final sequencing library as a result. For this reason, the hybrid capture method is a preferred
`
`methodfor enriching for desired sequences from a complex pool of nucleic acids andis ideal
`
`for preparing templates in next generation sequencing (NGS) applications.
`
`[05]
`
`The NGS applications usually involve randomly breaking long genomic DNA or
`
`cDNAinto smaller fragment sizes having a size distribution of 100-3,000 bp in length,
`
`depending upon the NGSplatform used. The DNA termini are enzymatically treated to
`
`facilitate ligation and universal DNA adaptors are ligated to the ends to provide the resultant
`
`NGStemplates. The terminal adaptor sequences provide a universal site for primer
`
`hybridization so that clonal expansionof the desired DNAtargets can be achieved and
`
`introduced into the automated sequencing processes used in NGSapplications. The hybrid
`
`capture method is intended to reduce the complexity of the pool of random DNA fragments
`
`from, for example, from 3 x 10° bases (the human genome) to much smaller subsets of 10° to
`
`10° bases that are enriched for specific sequencesofinterest. The efficiency of this process
`
`directly relates to the quality of capture and enrichment achieved for desired DNA sequences
`
`from the starting complex pool.
`
`[06]
`
`The NGSapplications typically use the hybrid capture method of enrichmentin the
`
`following manner. A prepared pool of NGS templates is heat denatured and mixed with a
`
`pool of capture probe oligonucleotides (“baits”). The baits are designed to hybridize to the
`
`regions of interest within the target genome and are usually 60-200 basesin length and
`
`further are modified to contain a ligand that permits subsequent capture of these probes. One
`
`common capture method incorporates a biotin group (or groups) on the baits. After
`
`hybridization is complete to form the DNA template:bait hybrids, capture is performed with a
`
`componenthavingaffinity for only the bait. For cxamplc, streptavidin-magnctic beads can
`
`be used to bind the biotin moiety of biotinylated-baits that are hybridized to the desired DNA
`
`targets from the pool of NGS templates. Washing removes unbound nucleic acids, reducing
`
`the complexity of the retained material. The retained material is then eluted from the
`
`magnetic beads and introduced into automated sequencing processes.
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`[07]
`
`Though DNA hybridization with the baits can be exquisitely specific, unwanted
`
`sequences remain in the enriched pool following complction of the hybrid capture
`
`method. The largest fraction of these unwanted sequencesis present due to undesired
`
`hybridization events between NGS templates having no complementarity to the baits and
`
`NGStemplates that do. Two types of undesired hybridizations arising in the hybrid capture
`
`method include the following sequences: (1) highly repetitive DNA elements that are found
`
`in endogenous genomic DNA;and (2) the terminal adaptor sequencesthat are engineered into
`
`each of the NGS templates of the pool.
`
`[08]
`
`The repetitive endogenous DNA elements, such as an Alu sequence or LINE
`
`sequence, present in one DNA fragment in the complex pool can hybridize to another similar
`
`element present in another unrelated DNA fragment. These fragments, which may originally
`
`derive from very different locations within the genome, become linked during the
`
`hybridization process of the hybrid capture method. If one of these DNA fragments
`
`represents a desired fragment that contains a bindingsite for a bait, the unwanted fragment
`
`will be captured along with the desired fragment. This class of unwanted NGS templates can
`
`be reduced by adding an excess of the repeat elements to the hybridization reaction. Most
`
`commonly, human Cot-1 DNAis added to the hybridization reaction, which binds Alu, LINE,
`
`and other repeat sites in the target and blocksthe ability of NGS templates to interact with
`
`each other on that basis.
`
`[09]
`
`<A more problematic class of unwanted NGStemplates that are recovered during
`
`hybrid capture arises from interactions between terminal adaptor sequences that are
`
`engineered on each of the NGS templates of the pool. Because the pool of NGS templates
`
`typically will contain the identical terminal adaptor sequences on every DNA fragment, the
`
`adaptor sequencesare present at a very high effective concentration(s) in the hybridization
`
`solution. Consequently, unrelated NGS templates can anneal to each other through their
`
`termini, thereby resulting in a “daisy chain” of otherwise unrelated DNA fragments being
`
`linked together. So if one of these linked fragments contains a binding site for a bait, the
`
`entire daisy chain is captured. In this way, capture of a single desircd fragment can bring
`
`along a large numberof undesired fragments, which reducesthe overall efficiency of
`
`enrichmentfor the desired fragment. This class of unwanted capture event can be reduced by
`
`adding an excess of single-stranded adaptor sequences to the hybridization reaction. Yet the
`
`ability to effectively reduce the so-called daisy chain capture events with an excess of adaptor
`
`sequencesis limited to an efficiency of about 50%-60% for capturing the desired fragment.
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`[10]
`
`In spite of the use of Cot-1 DNA and adaptor blocking oligonucleotides in the
`
`hybridization reaction, a significant amount of contaminating unwanted DNA fragments
`
`remain in the sequencing pool after the hybrid capture step, largely because the blocking
`
`methods are not completely successful. Thus, there is a need to improve capture efficiency
`
`and to reduce contamination from undesired sequences so that one can devote resources to
`
`sequencing a greater fraction of targets of interest and fewertargets that are not of interest.
`
`[11]
`
`Thus, off-target nucleic acid interactions can limit the efficiency of the selection of
`
`target nucleic acids by hybridization (for example, solution hybridization) to a capture probe,
`
`for example an oligonucleotide bait. Off-target selection can result, for example, in one or
`
`more of decreased yields of hybridization capture and/orartifactual hybrid capture, which in
`
`turn lead to inefficiencies in subsequentsteps, for example, sequencing.
`
`[12]
`
`Off-+target selection is typically increased whenthe stringency conditions of hybrid
`
`selection are reduced, for example, when selecting for a target:capture duplex having a lower
`
`nucleic acid melting temperature (for example, DNA:DNA duplexes as compared to
`
`RNA:DNAduplexes). Thus, capture of off-target sequence can be more of a problem in
`
`DNA:DNAhybridizations.
`
`[13]
`
`Typically, library members include a library insert, often a segment of sequence from
`
`a gene of interest, for example, a segment for sequencing. Ifa memberis on-target, the
`
`library insert forms a duplex with the capture probe. Typically, library members also include
`
`and one or more non-target sequences. These are typically not portions of a gene of interest
`
`but rather are adaptor sequences, amplification primers or tags, or bar code tags. The non-
`
`target sequence of the capture probe-hybridized library member, can, by duplex formation
`
`with other sequences in the reaction mixture, lead to the selection of undesired sequences, for
`
`example, off-target library members. While not wishing to be bound by theory,
`
`concatcnation between an on-target library memberand off-targct sequences can result in
`
`selection of off-target sequences.
`
`[14] Mcthods and compositions for minimizing selection of off-target nucleic acid, for
`
`example, minimizing the selection of library members that do not from a duplex with the
`
`capture probe are disclosed herein. Methods and compositions are disclosed herein that
`
`reduce non-target sequence, for example, adaptor-mediated selection.
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`BRIEF SUMMARYOF THE INVENTION
`
`[15]
`
`In one aspect, the invention relates to an oligonucleotide for use in a selection method
`
`of a desired template nucleic acid, comprising an oligonucleotide having at least one
`
`Tm-enhancing group. In first respect, the oligonucleotide is useful in selection methods such
`
`as the hybrid capture method. In second respect, the oligonucleotide includes as desired
`
`template nucleic acid at least one memberselected from a population of templates. In a third
`
`respect, the oligonucleotide is substantially complementary to at least one sequenceof the
`
`desired template. In a fourth respect, the oligonucleotide includes at least one member
`
`selected from a blockeror a bait. In a fifth respect, the oligonucleotide includesas the at least
`
`one Tm-enhancing group at least one memberselected from the group consisting of a locked
`
`nucleic acid group, a bicyclic nucleic acid group, a C5-modified pyrimidine, a peptide nucleic
`
`acid group and combinations thereof. In the sixth respect, the oligonucleotide includes as the
`
`at least one Tm-enhancing group one of a locked nucleic acid group, a bicyclic nucleic acid
`
`group or a combinationthereof. In a seventh respect, the oligonucleotide includesas the at
`
`least one Tm-enhancing group a locked nucleic acid group or a bicyclic nucleic acid group. As
`
`a preferred embodimentof the seventh respect, the oligonucleotide has as the locked nucleic
`
`acid group or the bicyclic nucleic acid group a nucleobaseselected from the group consisting
`
`of cytosine, adenine and thymine, including mixtures of cytosine and adenine and mixtures of
`
`cytosine and thymine. In a ninth respect, the oligonucleotide includes as the at least one Tm-
`
`enhancing group one that provides an optimal enhanced Tm value in the range comprising
`
`from about 1.4° C to about 25° C. In a tenth respect, the oligonucleotide includesat least one
`
`memberselected from the group consisting of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13,
`
`14, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 32, 34 and 36. In an eleventh respect,
`
`oligonucleotide includes a blocker. In a preferred embodiment of these respects, the blocker
`
`has substantial sequence complementarity to at least one sequence at a terminus of the desired
`
`template nucleic acid. In a further elaboration ofthis preferred embodiment, the blocker
`
`includes a barcode (or index) domain having a plurality of nucleotides. In a further
`
`embodimentof this respect, the plurality of nucleotides includes from about 5 to about 12
`
`nucleotides arranged substantially contiguous. In another embodiment, the barcode domain
`
`comprises nucleotides having as nucleobasesat least one memberselected from the group
`
`selected from adenine, thymine, cytosine, guanine, inosine, 3-nitropyrrole, 5-nitroindole, and
`
`combinationsthereof. In a twelfth respect, the oligonucleotide provides an improvement in
`
`the selection method of a desired template nucleic acid. In a preferred embodimentofthis
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`respect, the improvement consists of an improved enrichment of the desire template nucleic
`
`acid relative to undesired template nucleic acids. In yet another embodiment, the improved
`
`enrichment comprises of an enrichment of at least 65%. In the thirteenth respect, the
`
`oligonucleotide further includes a 3’-terminal modification. In this respect, preferred
`
`embodiments of the 3’-terminal modification prevents polymerase directed synthesis from the
`
`oligonucleotide. In another respect, the
`
`3’-terminal modification includes a 2’,3’-dideoxynucleotide, a 3’-spacer C3 group among
`
`others.
`
`[16]
`
`Inasecond aspect, the invention relates to a method of selecting a desired template
`
`nucleic acid from a population of template nucleic acids. The method includes two steps. The
`
`first step is contacting the population of template nucleic acids with a first oligonucleotide
`
`comprising a Tm-enhanced oligonucleotide to form a mixture. The second step includes
`
`isolating the desired template nucleic acid from the mixture. In a first respect, the method
`
`provides as part of the contacting step the sub-step of incubating the mixture at a temperature
`
`of about optimal enhanced Tm value of the Tm-enhanced oligonucleotide. In a first preferred
`
`embodimentof this respect, the Tm-enhanced oligonucleotide includes a plurality of
`
`Tm-enhancing groups. In this regard, the plurality of Tm-enhancing groups comprises from
`
`about 2 to about 25 Tm-enhancing groups. Further embodiments provide that the plurality of
`
`Tm-enhancing groups comprises locked nucleic acid groups or a bicyclic nucleic acid groups.
`
`Preferred aspects of these embodiments includefeatures of the locked nucleic acid groups or
`
`the bicyclic nucleic acid groups having nucleobases selected from the group consisting of
`
`cytosine, adenine and thymine. In a second respect, the method includes as the Tm-enhanced
`
`oligonucleotide at least one memberselected from the group consisting of SEQ ID NOS: 2, 3,
`
`4,5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 32, 34 and 36. Ina
`
`third respect, method provides the Tm-enhanced oligonucleotide that includes a blocker. In a
`
`first preferred embodimentofthis respect, the blocker has substantial sequence
`
`complementarity to at least one sequence at a terminus of each memberof the population of
`
`template nucleic acids. In yet another preferred embodiment, the blocker further includes a
`
`barcode domain having a plurality of nucleotides. In some embodiments, the plurality of
`
`nucleotides includes from about 5 to about 12 nucleotides arranged substantially contiguous.
`
`In other embodiments, the barcode domain includes nucleotides having as nucleobasesat
`
`least one memberselected from the group selected from adenine, thymine, cytosine, guanine,
`
`or a universal base, such as inosine, 3-nitropyrrole, 5-nitroindole, and combinations thereof.
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`In a third respect, the method has as the contacting step the objective of resulting in
`
`substantial inhibition of complex formation between the desired template nucleic acid and
`
`undesired template nucleic acids. In a fourth respect, the method includes as the step of
`
`isolating the desired template nucleic acid two additional steps. The first step is forming a
`
`hybrid complex between the desired nucleic acid and a second oligonucleotide. The second
`
`step is separating the hybrid complex from the mixture. With regard to this fourth respect, the
`
`second oligonucleotide includes a bait. In certain embodiments, the bait comprises a sequence
`
`having substantial sequence complementarity to a sequence within the desired template
`
`nucleic acid. In other embodiments, the bait comprises a plurality of Tm-enhancing groups. In
`
`yet other embodiments, the bait includes a covalent modification to enable selection of the
`
`hybrid complex. As part of these latter embodiments, the covalent modification is a
`
`biotinylated group. Yet other embodiments provide for the hybrid complex being contacted
`
`with a solid support immobilized with avidin or streptavidin.
`
`[17]
`
`Ina third aspect, the invention relates to a method of performing massively parallel
`
`sequencing. The method includes four steps. Thefirst step is preparing a library population of
`
`template nucleic acids. The secondstep is contacting the library population of template
`
`nucleic acids with at least one Tm-enhanced oligonucleotide as a blocker, a plurality of
`
`oligonucleotides as baits and Cot-1 DNA to form a mixture. The third step is isolating a
`
`plurality of desired template nucleic acids from the mixture. The fourth step is sequencing the
`
`plurality of desired template nucleic acids. The at least one member ofthe plurality of
`
`oligonucleotides as baits has substantial complementarity to a sequence within at least one
`
`memberof the plurality of desired template nucleic acids. In a first respect, the method
`
`includes membersof the library population of template nucleic acids each includesat least
`
`one identical terminal adaptor sequence having a size range from about 15 nucleotides to
`
`about 75 nucleotides. In a second respect, the method includes a blocker having substantial
`
`sequence complementarity to the at least one identical terminal adaptor sequence ofthe
`
`library population of template nucleic acids. In a third respect, the method includesas theat
`
`least one identical terminal adaptor sequence a barcode domain. In a fourth respect, the
`
`method provides a blocker having substantial sequence complementarity to the at least one
`
`identical terminal adaptor sequence. In a fifth respect, method includesas the contacting step
`
`the step of incubating the mixture at a temperature of about optimal enhanced Tm value of the
`
`at least one Tm-enhanced oligonucleotide. In a sixth respect, the method provides that the at
`
`least one Tm-enhanced oligonucleotide as a blocker includes at least one memberselected
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`from the group consisting of SEQ ID NOS: 2,3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 18,
`
`19, 21, 22, 24, 25, 27, 28, 30, 32, 34 and 36. In a seventh respect, method provides that the
`
`step of isolating a plurality of desired template nucleic acids from the mixture includes two
`
`steps. Thefirst step is forming a plurality of hybrid complexes between the plurality of
`
`desired template nucleic acids andplurality of oligonucleotides as baits. The secondstep is
`
`separating the plurality of hybrid complexes from the mixture. In an eighth respect, the
`
`methodprovidesas the plurality of oligonucleotides as baits includes a plurality of Tm-
`
`enhancing groups. In an embodiment ofthis respect, each bait includes a covalent
`
`modification to enable selection of the hybrid complex that includesthe bait. In a further
`
`embodiment of this respect, the covalent modification is a biotinylated group. As another
`
`embodimentof this respect, the plurality of hybrid complexes is contacted with a solid
`
`support immobilized with avidin or streptavidin.
`
`[18]
`
`In another aspect, the invention features, a method of selecting nucleic acids or of
`
`reducing off-target nucleic acid selection in hybridization reactions. The hybridization
`
`reaction can be a solid phase or solution phase hybridization. The method can be used in the
`
`selection of library members for subsequent processing, for example, for sequencing.
`
`[19]
`
`The method comprises:
`
`(a) optionally, acquiring a library comprising a plurality of target members, for
`
`example, target nucleic acid (for example, DNA or RNA) members, wherein one or more of
`
`the target members comprise an insert sequence (for example, a segment of a gene of interest)
`
`and a non-target nucleic acid sequence (for example, an adaptor sequence); and
`
`(b) contacting the library with a capture probe, for example, a bait set or a plurality of
`
`bait sets, and a blocking oligonucleotide,
`
`wherein,
`
`(i) a blocking oligonucleotide is complementary to, or can form a duplex with, the
`
`non-target nucleic acid sequenceof the library member (for example, an adaptor sequence),
`
`and
`
`(ii) the valuc for a parameter related to the binding intcraction between the blocking
`
`oligonucleotide and a non-target nucleic acid sequence ofthe library memberis higher than
`
`the value for the non-target nucleic acid sequence to a background nucleic acid, for example,
`
`other complementary non-target nucleic acid sequences,
`
`thereby minimizing off-target selection.
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`[20]
`
`In an embodiment the method further comprising providing selected library members
`
`(sometimesreferred to herein as “library catch”’).
`
`[21]
`
`In an embodiment, the method further comprises separating the selected library
`
`members from the capture probe.
`
`[22]
`
`In an embodiment, the method further comprises sequencing the insert of a selected
`
`library member, for example, sequencing the inserts from least 2, 5, 10, 15, 20, 30, or 50,
`
`genesor nucleic acid alterations, for example, genes or nucleic acid alterations described
`
`herein.
`
`[23]
`
`In an embodiment, the value for a parameterrelated to binding interaction can be a
`
`value for affinity, association rate, the inverse of dissociation rate, or nucleic acid melting
`
`temperature (for example, Tm, the temperature at which half of the DNA strands are in the
`
`double-helical state and half are in the random coilstate).
`
`[24]
`
`In an embodiment, the method comprises the use ofa first blocking oligonucleotide
`
`which forms a duplex with a first non-target nucleic acid sequence, for example, a first
`
`adaptor sequence, and, optionally, a second blocking oligonucleotide which forms a duplex
`
`with a second non-target nucleic acid sequence, for example, a second adaptor sequence. A
`
`set of oligonucleotide blockers comprises a plurality of different oligonucleotide blockers.
`
`[25]
`
`In an embodiment the blocking oligonucleotide inhibits the formation of a duplex
`
`between a sequence in the reaction and the non-target sequence ofa library memberthatis
`
`duplexed to the capture probe (for example, the blocking oligonucleotide inhibits formation
`
`of concatenated chains of library members).
`
`[26]
`
`In an embodiment, a library member comprises an insert, for example, a subgenomic
`
`interval, and a non-target sequence, for example, a sequence commonto a plurality of library
`
`members. In an embodiment, the inserts are subgenomic sequences, for example, from
`
`nucleic acid from a tumor sample, and the non-target sequence is non-genomically occurring
`
`sequence or a sequence not present in the subgenomic scquences, for example, an
`
`amplification tag or bar codingtag.
`
`[27]
`
`In an cmbodiment, the library members, or selected library members, include
`
`subgenomic intervals from at least 2, 5, 10, 15, 20, 30, or 50, genes or nucleic acid
`
`alterations, for example, genes or nucleic acid alterations described herein.
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`10
`
`[28]
`
`In an embodiment, a plurality of library members, or selected library members, for
`
`example, at X (whercin X is cqual to 2, 5, 10, 20, 50, 100, 200 or morc) library members, or
`
`selected library members, have a first non-target sequence at the 5’ end of the insert and a
`
`second non-target sequence at the 3’ end ofthe insert.
`
`[29]
`
`In an embodiment the non-target sequence includes a non-target sequencethat is
`
`present in a plurality of non-target sequences, for example, a sequence for amplification, and
`
`a non-target sequence that is unique, for example, a barcode. Typically some, most
`
`substantially all or all of the membersof the library will include a commonnon-target
`
`sequence. In embodiments the library, or the selected library members, comprises at least X
`
`members, (wherein X is equal to 1, 2, 5, 10, 20, 50, 100, 200 or more) having a common non-
`
`target sequence.
`
`[30]
`
`In one embodiment, the blocking oligonucleotide forms a duplex with a non-target
`
`nucleic acid sequence of at least X library members (wherein X is equal to 1, 2, 5, 10, 20, 50,
`
`100, 200 or more), which duplex has a Tm that is higher than the Tm of a duplex formed by a
`
`non-target nucleic acid sequence to a background nucleic acid, for example, the complement
`
`of the non-target sequence. In one embodiment, the higher nucleic acid melting temperature
`
`of the modified blocking oligonucleotide duplex is from about 5°C to about 25°C higher than
`
`that of an unmodified blocking oligonucleotide duplex, or greater (for example,
`
`2° C, 5° C, 10° C, 15° C, 20° C, 25° C, or greater). In one embodiment, the Tm for the duplex
`
`between the blocking oligonucleotide and the non-target nucleic acid sequence ofthe library
`
`memberis higher than is the Tm for a duplex of the non-target nucleic acid sequence andits
`
`exact complement.
`
`[31]
`
`In other embodiments, the blocking oligonucleotide has an association rate to a non-
`
`target nucleic acid sequence of at least X library members (wherein X is equal to 1, 2, 5, 10,
`
`20, 50, 100, 200 or more), that is higher than the association rate of the non-target nucleic
`
`acid sequence to a background nucleic acid, for example, the complement of the non-target
`
`sequence. In one embodiment, the higher association rate is about 2- to greater than 10-fold
`
`that of the non-target nucleic acid sequence to the background nucleic acid (for example, 2-,
`
`4., 6-, 8-, 10-fold, or greater).
`
`[32]
`
`In yet other embodiments, the blocking oligonucleotide has a dissociation rate to the
`
`non-target nucleic acid sequence of at least X library members (wherein X is equal to 1, 2, 5,
`
`10, 20, 50, 100, 200 or more) that is lower than the dissociation rate of the non-target nucleic
`
`

`

`WO 2014/008447
`
`PCT/US2013/049402
`
`11
`
`acid sequence to a background nucleic acid, for example, the complement of the non-target
`
`sequence. In one embodiment, the lower dissociation rate is about 2- to greater than 10-fold
`
`that of the non-target nucleic acid sequence to the background nucleic acid (for example, 2-,
`
`4., 6-, 8-, 10-fold, or greater).
`
`[33]
`
`In one embodiment, the length of the blocking oligonucleotide results in an increase
`
`in the binding interaction of the blocking oligonucleotide for the non-target nucleic acid
`
`sequence ofthe library member(for example, the adaptor sequence), relative to the
`
`background nucleic acid.
`
`[34]
`
`In an embodiment, the duplex formed between the blocking oligonucleotide and non-
`
`target nucleic acid sequence of at least X library members (wherein X is equal to 1, 2, 5, 10,
`
`20, 50, 100, 200 or more), is longer than the duplex formed between the non-target sequence
`
`and its complement, for cxamplce, between the Watson and Crick strands of a double-stranded
`
`adaptor. In embodiments, the duplex between a blocking oligonucleotide and non-target
`
`nucleic acid sequenceis at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, or 20 nucleotides longer than
`
`the duplex formed between the non-target sequence and its complement, for example,
`
`between the Watson and Crick strands of a double-stranded adaptor.
`
`[35]
`
`In an embodiment, the blocking oligo comprises one or more non-naturally-occurring
`
`nucleotides. In embodiments a duplex formed betweenthe blocking oligonucleotide having
`
`non-naturally-occurring nucleotides and the non-target nucleic acid sequence ofat least X
`
`library members (wherein X is equal to 1, 2, 5, 10, 20, 50, 100, 200 or more), has the value
`
`for a parameter related to the binding interaction (for example, affinity, association rate,
`
`inverse of dissociation rate, or Tm) that is higher than the value for the non-target nucleic acid
`
`sequence to a background nucleic acid, for example, other complementary non-target nucleic
`
`acid sequences. Exemplary non-naturally occurring oligonucleotides include modified DNA
`
`or RNA nuc