(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`19 September 2013 (19.09.2013)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
`
`WO 2013/138536 A1
`
`(51)
`
`International Patent Classification:
`CIZQ 1/68 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2013/031104
`
`(22)
`
`International Filing Date:
`
`13 March 2013 (13.03.2013)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`(81)
`
`Filing Language:
`
`Publication Language:
`
`English
`
`English
`
`Priority Data:
`61/610,296
`61/613,784
`
`13 March 2012 (13.03.2012)
`21 March 2012 (21.03.2012)
`
`US
`US
`
`Applicant: SWIFT BIOSCIENCES, INC. [US/US]; 58
`Parkland Plaza, Suite 100, Ann Arbor, MI 48103 (US).
`
`Inventors: MAKAROV, Vladimir; 1601 Pond Shore
`Drive, Ann Arbor, MI 48108 (US). KURIHARA, Laurie;
`2756 Holyoke Lane, Ann Arbor, MI 48103 (US).
`
`Agents: ALLIKIAN, Michael J. et a1.; Marshall, Gerstein
`& Borun LLP, 233 S. Wacker Drive, 6300 Willis Tower,
`Chicago, IL 60606-6357 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`
`A0, AT, AU, Az, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CII, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`IIN, IIR, IIU, ID, IL, IN, IS, JP, KE, KG, KM, 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,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG).
`Published:
`
`with international search report (Art. 21(3))
`
`with sequence listing part ofdescription (Rule 5.2(a))
`
`(54) Title: METHODS AND COMPOSITIONS FOR SIZE-CONTROLLED HOMOPOLYMER TAILING OF SUBSTRATE
`POLYNUCLEOTIDES BY A NUCLEIC ACID POLYMERASE
`
`
`
`
`
`SIDZ'VU'9Zt‘OI1951511"le
`
`DNA primer
`5' —’ 3'
`
`TdT, dATP
`
`Attenuator (dT)30
`3'PedeTdeTdeTdeTdeTdeTdeTdeTdTdeTdTdeTdeTdeTdeTdeTdTr5'
`
`UTailed DNA primer
`5' — dAdAdAdAdAdAdAdAdA-3'
`
`Attenuator (dT)30
`G'P-deTdeTdeTdeTdeTdTdeTdeTdTdTdTdTdeTdeTdeTdeTdeTdeT—S'
`
`TdT, dATP
`
`fl
`Tailed DNA primer - Attenuator (dT)30complex with Tm = or > 37“C
`5' — d/‘ldflltdA‘dAldAdlAdlAdlAdlAdlAdlldeldAIdA-IE'
`3 P-deTdeTdeTd TdeTde TdeTdeTdeTdeTdeTd TdeTdeTdeT-S
`
`Figure 1
`
`(57) Abstract: The present inVention is directed to methods and compositions for adding tails of specific lengths to a substrate poly—
`nucleotide. The invention also contemplates methods and compositions for immobilization of tailed substrates to a solid support.
`The disclosure contemplates that the attenuator molecule is any biomolecule that associates With a tail sequence added to a substrate
`polynucleotide and controls the addition of a tail sequence to the 3' end of the substrate polynucleotide. The sequence that is added
`to the substrate polynucleotide is referred to herein as a tail sequence, or simply a tail, and the process of adding a nucleotide to a
`substrate polynucleotide is referred to herein as tailing.
`
`
`
`W02013/138536A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`METHODS AND COMPOSITIONS FOR SIZE-CONTROLLED HOMOPOLYMER
`TAILING OF SUBSTRATE POLYNUCLEOTIDES BY A NUCLEIC ACID
`POLYMERASE
`
`CROSS REFERENCE TO RELATED APPLICATIONS
`
`[0001]
`
`This application claims the priority benefit under 35 U.S.C. § 119(e) of US.
`
`Provisional Application No. 61/610,296, filed March 13, 2012, and US. Provisional Application
`
`No. 61/613,784, filed March 21, 2012, each of which is incorporated herein by reference in their
`
`entirety.
`
`FIELD OF THE INVENTION
`
`[0002]
`
`The present invention is directed to methods and compositions for adding tails of
`
`specific lengths to a substrate polynucleotide. The invention also contemplates methods and
`
`compositions for immobilization of tailed substrates to a solid support.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Many current next—generation sequencing (NGS) platforms require special DNA and
`
`RNA preparations prior to sequencing. Most commonly used preparations involve addition of
`
`adaptor sequences to the ends of double—stranded DNA fragments through a ligation reaction.
`
`The reaction typically involves blunt—ended DNA or DNA with a single deoxyadenosine (dA)
`
`nucleotide at the 3' end and a high concentration of DNA ligase, and the reaction results in
`
`formation of a significant number of chimeric templates. Template—independent polymerases
`
`such as DNA—specific terminal deoxynucleotidyl transferase (TdT), and RNA—specific poly(A)
`
`and poly(U) polymerases potentially represent an attractive alternative approach for preparation
`
`of DNA and RNA for NGS analysis with the challenging caveat that the length of polymeric tails
`
`produced by these enzymes varies in a wide range (from 20 to 500 nucleotides), depends on
`
`many factors, and is not easy to control, thus reducing their utility for NGS.
`
`SUMMARY OF THE INVENTION
`
`[0004] Accordingly, the present disclosure provides a composition comprising a nucleic acid
`
`polymerase and an attenuator molecule. The disclosure contemplates that the attenuator
`
`molecule is any biomolecule that associates with a tail sequence added to a substrate
`
`polynucleotide and controls the addition of a tail sequence to the 3' end of the substrate
`
`polynucleotide. The sequence that is added to the substrate polynucleotide is referred to herein
`
`1
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`as a tail sequence, or simply a tail, and the process of adding a nucleotide to a substrate
`
`polynucleotide is referred to herein as tailing. An attenuator molecule, as used herein, is a
`
`polynucleotide, a polypeptide, a polysaccharide, and combinations thereof.
`
`In aspects Where the
`
`attenuator molecule is a polynucleotide, it is further contemplated that the polynucleotide is a
`
`circular molecule, or that the polynucleotide comprises a peptide nucleic acid, a Schizophyllan
`
`polysaccharide, a locked nucleic acid and combinations thereof.
`
`[0005] As described above, the attenuator molecule associates with a tail sequence added to
`
`the substrate polynucleotide and controls the addition of nucleotides thereto. In some
`
`embodiments, the attenuator molecule is a polynucleotide that hybridizes to a sequence added to
`
`a substrate polynucleotide, wherein the number of nucleotides added to the substrate
`
`polynucleotide is essentially equal to the number of nucleotides in the portion of the attenuator
`
`molecule that associates with the tail sequence.
`
`In some aspects, the number of nucleotides
`
`added to the substrate polynucleotide is essentially equal to a multiple of the number of
`
`nucleotides in the attenuator molecule that associates With the tail sequence. As used herein, the
`
`terms "essentially" and "essentially equal" are understood to mean approximately or
`
`approximately equal.
`
`[0006]
`
`In some embodiments, the nucleic acid polymerase is a template—independent
`
`polymerase. In one aspect, the nucleic acid polymerase is a DNA polymerase, and in a further
`
`aspect the DNA polymerase is terminal deoxynucleotidyl transferase (TdT). In related
`
`embodiments, the nucleic acid polymerase is a RNA polymerase, which in various aspects is
`
`selected from the group consisting of poly(A) polymerase, RNA-specific nucleotidyl transferase
`
`and poly(U) polymerase.
`
`[0007]
`
`It is contemplated by the disclosure that, in some embodiments, the attenuator
`
`molecule comprises a nucleotide selected from the group consisting of 2'—deoxythymidine 5'—
`
`monophosphate (dTMP), 2'—deoxyguanosine 5'-monophosphate (dGMP), 2'-deoxyadenosine 5'-
`
`monophosphate (dAMP), 2'-deoxycytidine 5'—monophosphate (dCMP), 2'-deoxyuridine 5’—
`
`monophosphate (dUMP), thymidine monophosphate (TMP), guanosine monophosphate (GMP),
`
`adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate
`
`(UMP), a base analog, and combinations thereof. Thus, in certain embodiments the attenuator
`
`molecule comprises an attenuator sequence that is a heteropolymeric sequence or a
`
`2
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`homopolymeric sequence, wherein the sequence is either a dinucleotide sequence or a
`
`homopolymer sequence.
`
`[0008]
`
`In various aspects, the attenuator molecule comprises 1 nucleotide, 2 nucleotides, 3
`
`nucleotides, 4 nucleotides, 5 nucleotides, 10 nucleotides, 20 nucleotides, 30 nucleotides, 50
`
`nucleotides, 100 nucleotides or more.
`
`[0009]
`
`The attenuator molecule, in some embodiments, comprises a blocking group and in one
`
`aspect, the blocking group is on the 3' end of the molecule. The blocking group prevents
`
`extension of the attenuator molecule by the nucleic acid polymerase. Thus, in various aspects
`
`the blocking group is selected from the group consisting of at least one ribonucleotide, at least
`
`one deoxynucleotide, a C3 spacer, a phosphate, a dideoxynucleotide, an amino group, and an
`
`inverted deoxythymidine.
`
`[0010]
`
`In some embodiments of the disclosure, the attenuator molecule further comprises an
`
`adaptor sequence, an identifier tag sequence, or both, located 5' to the attenuator sequence
`
`homopolymer sequence or dinucleotide sequence.
`
`In one embodiment, the attenuator molecule
`
`is immobilized.
`
`In further embodiments, a ligase is present in the composition. In another
`
`embodiment, the composition further comprises a ligase enzyme.
`
`[0011]
`
`In another embodiment, the attenuator molecule is an attenuator—adaptor molecule
`
`which comprises an attenuator sequence and further comprises a sequence W positioned adjacent
`
`the attenuator sequence and is complementary to a sequence X on a separate polynucleotide; the
`
`composition further comprising an adaptor molecule comprising a sequence Y complementary to
`
`a sequence V, wherein sequence V is the same length as Y or is less than the same length as
`
`sequence Y, the adaptor molecule being a separate molecule from the attenuator—adaptor
`
`molecule.
`
`[0012]
`
`In some embodiments of the disclosure, the attenuator molecule further comprises a
`
`next generation sequencing (NGS) adaptor sequence. An NGS adaptor sequence differs from an
`
`adaptor sequence in that the NGS adaptor sequence is useful in a sequencing platform. In some
`
`embodiments of the disclosure, the attenuator molecule further comprises a next generation
`
`sequencing (NGS) adaptor sequence comprises sequence X and sequence Y (for further
`
`description of the various sequences (e.g., sequence X, sequence Y, etc.) discussed herein, see
`
`Figures and discussion, below), an identifier tag sequence, or a combination thereof, located 5' to
`
`3
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`a homopolymer sequence or dinucleotide sequence. In embodiments wherein the attenuator
`
`molecule further comprises an adaptor sequence, it is referred to herein as an attenuator—adaptor
`
`molecule. In one embodiment, the attenuator molecule and/or attenuator—adaptor molecule is
`
`immobilized. In related embodiments, sequence X and sequence Y are NGS adaptor sequences
`
`that are compatible with Illumina, Ion Torrent, Roche 454 or SOLiD sequencing platforms.
`
`[0013]
`
`In further embodiments, sequence X and sequence Y are on separate attenuator-adaptor
`
`molecules, while in other embodiments sequence X and sequence Y are adjacent to each other on
`
`the same attenuator-adaptor molecule.
`
`[0014]
`
`The disclosure also provides, in some embodiments, compositions wherein an adaptor
`
`sequence further comprises a cleavable sequence (Z) that is located between sequence X and
`
`sequence Y. In some aspects, the Cleavage site in sequence Z is at least one dU base or RNA
`
`base, or a restriction endonuclease site.
`
`[0015] Also provided by the disclosure are compositions wherein the attenuator molecule is
`
`single stranded or is at least partially double stranded. By "partially double stranded" is meant
`
`that the attenuator molecule comprises a single stranded portion and a double stranded portion.
`
`In some aspects wherein the attenuator molecule is at least partially double stranded, the partially
`
`double stranded attenuator molecule is produced by annealing a portion of the attenuator
`
`molecule to an adaptor molecule (which comprises an adaptor sequence that, in some
`
`embodiments, is an NGS adaptor sequence) to which it is complementary. In some aspects,
`
`annealing is (a) between an attenuator molecule and a separate adaptor molecule, or (b)
`
`annealing occurs in a single attenuator molecule that forms a hairpin structure, thus the
`
`attenuator molecule comprises both a homopolymeric sequence or dinucleotide sequence and an
`
`adaptor sequence.
`
`[0016]
`
`In still further embodiments, the attenuator molecule comprises a sequence W that is
`
`fully complementary to adaptor sequence X.
`
`In some aspects, sequence W is also all or partially
`
`complementary to adaptor sequence Y.
`
`[0017]
`
`In various aspects, the attenuator molecule comprises a homopolymeric sequence
`
`selected from the group consisting of poly (dA), poly (dT), poly (dC), poly (dG), poly (dU), poly
`
`(rA), poly (U), poly (rC), poly (rG) and a heteropolymeric, or a dinucleotide, sequence
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`comprising combinations of:
`
`(i) dA and rA bases, (ii) dT, dU and U bases, (iii) dC and 1C bases,
`
`or (iv) dG and rG bases.
`
`[0018]
`
`In further aspects, the attenuator molecule comprises deoxyribonucleotides and is
`
`degradable with a DNA—specific nuclease.
`
`In some of these aspects, the DNA—specific nuclease
`
`is DNase I. In further embodiments, a composition provided by the disclosure comprises a
`
`single strand circularization ligase, including but not limited to CircLigase and/or CircLigase II.
`
`[0019]
`
`The disclosure also provides, in some aspects, a composition that comprises a DNA
`
`polymerase which lacks proofreading activity, and Kapa HiFi Polymerase, which possesses
`
`proofreading activity. In additional embodiments, a composition of the disclosure comprises a
`
`ligase enzyme.
`
`[0020]
`
`Further embodiments of the disclosure provide a composition that comprises a
`
`restriction endonuclease capable of cleaving sequence Z, which is located between sequence X
`
`and sequence Y and comprises a restriction endonuclease site, when hybridized to a
`
`complementary X'Z'Y' polynucleotide.
`
`[0021]
`
`In some embodiments, a composition is provided wherein a partially double stranded
`
`adaptor sequence comprised of sequence V and sequence Y wherein V is a truncated
`
`complement of Y and comprises a blocked 3' end, such that the partially double stranded adaptor
`
`can be blunt ligated to a double stranded substrate molecule. In another embodiment, sequence
`
`V is fully complementary to sequence Y.
`
`[0022]
`
`The disclosure further contemplates compositions wherein the attenuator molecule is
`
`degradable. In some aspects, the attenuator molecule comprises dU bases and is degradable by
`
`incubation with a dU—glycosylase (which creates abasic sites) followed by incubation at a
`
`temperature that is above 80° C (introduces breaks within abasic sites), or a mixture of dU—
`
`glycosylase and an apurinic/apyrimidinic endonuclease. Thus, the disclosure provides
`
`compositions, in various aspects, wherein the attenuator molecule comprises dU bases and
`
`incubation with a dU—glycosylase destabilizes the attenuator molecule, or incubation with a dU—
`
`glycosylase and subsequent incubation at a temperature that is above 80° C degrades the
`
`attenuator molecule, or the attenuator molecule is incubated with a mixture of dU—glycosylase
`
`and an apurinic/apyrimidinic endonuclease. In further aspects, the attenuator molecule
`
`comprises a ribonucleotide and is degradable with a ribonuclease under conditions sufficient for
`5
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`ribonuclease activity.
`
`In related aspects, the ribonuclease is selected from the group consisting
`
`of RNase H, RNase HII, RNase A, and RNase T1.
`
`[0023]
`
`In further aspects, the attenuator molecule comprises deoxyribonucleotides and is
`
`degradable with a DNA—specific nuclease.
`
`In some of these aspects, the DNA—specific nuclease
`
`is DNase I.
`
`[0024]
`
`The disclosure also provides a method of extending a substrate polynucleotide
`
`comprising incubating the substrate polynucleotide with a composition as described herein under
`
`conditions sufficient to allow addition of a tail sequence to the 3' end of the substrate
`
`polynucleotide, and wherein the addition of the tail sequence allows association between the tail
`
`sequence and the attenuator molecule to form a complex. In another aspect, the method further
`
`comprises degrading the attenuator molecule following extension of the substrate polynucleotide.
`
`In a further aspect, the method further comprises isolating the extended substrate polynucleotide.
`
`Other aspects of the methods further comprise mixing a composition as described herein with the
`
`substrate polynucleotide and a nucleotide that is complementary to the homopolymeric portion of
`
`the attenuator molecule.
`
`[0025] According to various aspects of the disclosure, the substrate polynucleotide is a single
`
`stranded polynucleotide or is a double stranded polynucleotide. The double stranded
`
`polynucleotide, in some aspects, has a blunt end, a 3' overhanging end, a 3' recessed end, or a
`
`free 3' hydroxyl group. The present disclosure provides methods wherein the substrate
`
`polynucleotide is double stranded, and in certain aspects, the double stranded substrate
`
`polynucleotide is produced by annealing a first substrate polynucleotide to a second substrate
`
`polynucleotide under conditions sufficient to allow the first substrate polynucleotide to associate
`
`with the second substrate polynucleotide. According to further aspects of the disclosure, the
`
`substrate polynucleotide comprises a free 3' hydroxyl group. The single stranded polynucleotide,
`
`in various embodiments, is prepared by denaturation of fragmented double stranded DNA or
`
`from reverse transcription of RNA. The double stranded polynucleotide, in some aspects, has a
`
`blunt end or a 3' overhanging end with a free 3' hydroxyl group.
`
`[0026]
`
`In various aspects of the methods of the disclosure, a multiplicity of nucleotides are
`
`added to the substrate polynucleotide. The number of nucleotides added to the substrate
`
`polynucleotide comprises, in various aspects, at least about 1 nucleotide and up to about 10, 20,
`
`6
`
`

`

`WO 2013/138536
`
`PCT/US2013/031104
`
`50 or 100 nucleotides, at least about 3 nucleotides and up to about 10, 20, 50 or 100 nucleotides,
`
`at least about 10 nucleotides and up to about 20, 30, 50 or 100 nucleotides, at least about 5
`
`nucleotides and up to about 10, 20, 50 or 100 nucleotides, at least about 10 nucleotides and up to
`
`about 20, 30, 50 or 100 nucleotides, at least about 1 nucleotide and up to about 5, 10, or 20
`
`nucleotides, at least about 3 nucleotides and up to about 5, 10, or 20 nucleotides, at least about 5
`
`nucleotides and up to about 20, 40 or 50 nucleotides, at least 1 nucleotide, at least 2 nucleotides,
`
`at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6, at least 7, at least 8,
`
`at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least
`
`17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at
`
`least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least
`
`34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at
`
`least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least
`
`51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at
`
`least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least
`
`68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at
`
`least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least
`
`85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at
`
`least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100 nucleotides or
`
`more.
`
`[0027]
`
`Further embodiments provided by the disclosure include those wherein the attenuator
`
`molecule associates With the tail sequence over all or a portion of the attenuator molecule length.
`
`In some embodiments, the attenuator molecule associates with the tail sequence during the
`
`process of adding the tail sequence. Association of the attenuator molecule to the tail sequence,
`
`in further aspects, regulates addition of nucleotides to the substrate polynucleotide. The
`
`attenuator molecule additionally comprises an adaptor sequence, in some aspects, and the
`
`adaptor sequence is ligated by a ligase enzyme to the substrate polynucleotide during addition of
`
`the tail sequence to the substrate polynucleotide, and in various embodiments the ligase is a
`
`DNA ligase or a RNA ligase.
`
`[0028]
`
`It is also contemplated by the disclosure that the conditions of the method, in some
`
`aspects, regulate addition of the tail sequence to the substrate polynucleotide. With respect to the
`
`conditions that are contemplated to regulate addition of the tail sequence to the substrate
`7
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`polynucleotide, it is contemplated that, in some aspects, the addition of the tail sequence to the
`
`substrate polynucleotide is temperature sensitive. Conditions wherein the temperature is
`
`between about 4° C and about 50° C are contemplated. In aspects wherein a thermostable
`
`polymerase is used, the temperature can be above 50° C. Accordingly, in further aspects the
`
`temperature is between about 50° C and about 90° C.
`
`[0029]
`
`In another embodiment, the addition of the tail sequence to the substrate
`
`polynucleotide is time sensitive, and in various aspects the incubation step is allowed to progress
`
`for a length of time in the range of about 0.5 minutes to about 120 minutes. In further
`
`embodiments, the addition of the tail sequence to the substrate polynucleotide is pH sensitive. In
`
`some of these embodiments, the addition of the tail sequence to the substrate polynucleotide is
`
`performed under conditions wherein pH is in the range of about pH 5.0 to about pH 9.0.
`
`[0030]
`
`In various embodiments, the substrate polynucleotide is DNA or RNA.
`
`[0031] Methods provided herein also include those wherein the attenuator—adaptor molecule is
`
`immobilized. In some aspects, the immobilized attenuator—adaptor molecule is ligated by a DNA
`
`or RNA ligase to a substrate polynucleotide during addition of a tail sequence to the substrate
`
`polynucleotide resulting in immobilization of the substrate polynucleotide. In further aspects,
`
`the amount of ligase enzyme added to a reaction is from about 0.1 to about 1000 units (U).
`
`[0032]
`
`In certain aspects, the methods described herein further comprise magnesium in an
`
`amount of about 1 mM to about 100 mM. In further aspects, the methods further comprise
`
`potassium or sodium in an amount of about 1 mM to about 1 M.
`
`[0033]
`
`In a specific aspect of the disclosure, a method of extending a DNA substrate
`
`polynucleotide is provided comprising mixing the DNA substrate polynucleotide with TdT
`
`enzyme, a degradable attenuator polynucleotide comprising a 3' phosphate and nucleotides that
`
`are complementary to the homopolymeric portion of the attenuator polynucleotide, incubating
`
`the mixture at 37° C for about 30 minutes, followed by an additional incubation at 70° C for
`
`about 10 minutes, degrading the attenuator molecule by adding a DNA glycosylase and
`
`incubating the mixture at 37° C for about 5 minutes, and optionally isolating the extended DNA
`
`substrate polynucleotide.
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`[0034]
`
`In another aspect, the disclosure provides a method of extending a DNA substrate
`
`polynucleotide comprising mixing a substrate polynucleotide with TdT enzyme, an attenuator
`
`polynucleotide comprising two ribonucleotides at the 3' end, and nucleotides that are
`
`complementary to the homopolymeric portion of the attenuator polynucleotide, incubating the
`
`mixture at 30°C for 30 minutes, followed by an additional incubation at 70°C for about 10
`
`minutes to inactivate the TdT enzyme, and optionally isolating the extended DNA substrate
`
`polynucleotide.
`
`[0035]
`
`The disclosure further provides, in one aspect, a method of extending a substrate RNA
`
`polynucleotide comprising mixing the substrate RNA polynucleotide with an RNA polymerase, a
`
`degradable attenuator polynucleotide and ribonucleotides that are complementary to the
`
`homopolymeric portion of the attenuator polynucleotide, incubating the mixture at 30° C for
`
`about 30 minutes, followed by an additional incubation at 95° C for about 10 minutes, degrading
`
`the attenuator molecule by adding a DNA glycosylase and incubating the mixture at 37° C for
`
`about 10 minutes, and optionally isolating the extended substrate polynucleotide.
`
`[0036]
`
`In another aspect, the disclosure provides a method of extending a DNA substrate
`
`polynucleotide comprising annealing attenuator and adaptor molecules that are partially
`
`complementary to each other by heating a mixture of the attenuator and the adaptor molecules in
`
`a suitable buffer to about 100° C and then cooling to about 25° C, wherein the annealing results
`
`in a partially double stranded attenuator—adaptor molecule, mixing the DNA substrate
`
`polynucleotide with TdT enzyme, a ligase enzyme, the partially double stranded attenuator—
`
`adaptor molecule and nucleotides that are complementary to the homopolymeric portion of the
`
`attenuator—adaptor molecule, incubating at about 37° C for about 15 to about 30 minutes, and
`
`optionally isolating the extended DNA substrate polynucleotide ligated to the attenuator—adaptor
`
`molecule.
`
`[0037]
`
`The disclosure further provides, in one aspect, a method of extending a DNA substrate
`
`polynucleotide comprising annealing one attenuator—adaptor molecule to a second, biotinylated
`
`adaptor molecule that are at least partially complementary to each other by heating a mixture of
`
`the two molecules in a suitable buffer to about 100° C and then cooling to about 25° C, wherein
`
`the annealing results in a double stranded biotinylated attenuator—adaptor molecule, immobilizing
`
`the double stranded biotinylated attenuator—adaptor molecule by mixing the double stranded
`
`9
`
`

`

`WO 2013/138536
`
`PCT/USZOl3/031104
`
`biotinylated attenuator—adaptor molecule with a solution comprising a streptavidin—coated
`
`magnetic bead at about 25° C for about 30 to about 60 minutes, resulting in immobilization of
`
`the double stranded biotinylated attenuator—adaptor molecule to the streptavidin—coated magnetic
`
`bead, incubating the immobilized double stranded biotinylated attenuator—adaptor molecule
`
`attached to the streptavidin—coated magnetic bead with the DNA substrate polynucleotide, TdT
`
`enzyme, a ligase enzyme and nucleotides that are complementary to the homopolymeric portion
`
`of the double stranded attenuator molecule at about 37°C for 15 to about 30 minutes, washing the
`
`solution with NaOH to remove non—biotinylated single stranded DNA from the beads, and
`
`optionally isolating the extended DNA substrate polynucleotide ligated to the double stranded
`
`biotinylated attenuator—adaptor molecule.
`
`[0038]
`
`In another aspect, a method of extending a RNA substrate polynucleotide is provided
`
`comprising annealing an attenuator molecule and an adaptor molecule that are at least partially
`
`complementary to each other by heating a mixture of the two molecules in a suitable buffer to
`
`about 100° C and then cooling to about 25° C, wherein the annealing results in a partially double
`
`stranded attenuator—adaptor molecule; mixing an RNA substrate polynucleotide with poly (A) or
`
`poly(U) enzyme, a ligase enzyme, the partially double stranded attenuator-adaptor molecule and
`
`ribonucleotides that are complementary to the single-stranded homopolymeric portion of the
`
`partially double stranded attenuator molecule; incubating at about 30° C to about 37° C for about
`
`15 — 30 minutes; and optionally isolating the extended RNA substrate polynucleotide ligated to
`
`the attenuator—adaptor molecule.
`
`[0039]
`
`In a further aspect, the disclosure provides a method of extending and immobilizing an
`
`RNA substrate polynucleotide comprising annealing an attenuator—adaptor molecule to a
`
`biotinylated adaptor molecule that are at least partially complementary to each other by heating a
`
`mixture of the two molecules in a suitable buffer to about 100° C and then cooling to about 25°
`
`C, wherein the annealing results in a partially double stranded biotinylated attenuator—adaptor
`
`molecule; immobilizing the partially double stranded biotinylated attenuator—adaptor molecule
`
`by mixing the partially double stranded biotinylated attenuator—adaptor molecule with a solution
`
`comprising a streptavidin—coated magnetic bead at about 25° C for about two hours, resulting in
`
`immobilization of the partially double stranded biotinylated attenuator-adaptor molecule to the
`
`streptavidin—coated magnetic bead; incubating the immobilized partially double stranded
`
`10
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`biotinylated attenuator—adaptor molecule attached to the streptavidin—coated magnetic bead with
`
`the RNA substrate polynucleotide, poly(A) or poly(U) polymerase, a ligase enzyme and
`
`ribonucleotides that are complementary to the single stranded homopolymeric portion of the
`
`partially double stranded attenuator—adaptor molecule at about 30° C to about 37° C for about 15
`
`— 30 minutes; washing the solution with N aOH to remove non—biotinylated single stranded
`
`polynucleotide from the beads; and optionally isolating the extended and immobilized RNA
`
`substrate polynucleotide ligated to the double stranded biotinylated attenuator—adaptor molecule.
`
`[0040]
`
`The disclosure also provides, in various embodiments, methods wherein a DNA
`
`polymerase and dNTPs are mixed to perform a polymerase extension of a substrate
`
`polynucleotide, said polymerase extension occurring subsequent to controlled homopolymer
`
`tailing and leading to incorporation of NGS adaptor sequence(s) sequence X, sequence Y or
`
`sequences X and Y 3' to the substrate homopolymer that are complementary to the additional
`
`sequence X' and sequence Y' that are 5' to the homopolymer of the attenuator molecule.
`
`[0041]
`
`In further embodiments, NGS adaptor sequence X, sequence Y or sequences X and Y
`
`are optionally ligated by a ligase enzyme to the substrate polynucleotide during addition of
`
`nucleotides to the substrate polynucleotide. In other embodiments, NGS adaptor sequence X,
`
`sequence Y or sequences X and Y are optionally ligated by a ligase enzyme to the substrate
`
`polynucleotide after addition of nucleotides to the substrate polynucleotide.
`
`In related
`
`embodiments, the ligase is a DNA ligase or a RNA ligase.
`
`[0042]
`
`In still further embodiments, an attenuator molecule sequence W is optionally
`
`truncated with respect to sequences X' and Y' to allow a full—length X'Y' polynucleotide primer
`
`to displace the truncated attenuator and enable polymerase extension to create a double stranded
`
`adapted substrate molecule. As used herein, an "adapted molecule" is a substrate molecule that
`
`has undergone a tailing and ligation reaction.
`
`[0043]
`
`The disclosure further provides embodiments wherein the substrate molecule,
`
`following homopolymer addition and polymerase extension, is optionally incubated with a single
`
`stranded DNA circularization ligase that results in circularization of the adapted single stranded
`
`DNA molecule. In one embodiment, circularization of the attenuator molecule comprising
`
`sequence X and sequence Y is prevented by degradation.
`
`ll
`
`

`

`WO 2013/138536
`
`PCT/U82013/031104
`
`[0044]
`
`In another embodiment, the substrate molecule, following homopolymer addition and
`
`ligation, is optionally incubated with a single stranded DNA circularization ligase which results
`
`in circularization of the adapted single stranded DNA molecule.
`
`[0045]
`
`In a further embodiment of the disclosure, circularization of the XZY adaptor molecule
`
`is prevented by formation of