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WO2025226876A1 - Procédés de normalisation de bibliothèque de séquençage - Google Patents

Procédés de normalisation de bibliothèque de séquençage

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Publication number
WO2025226876A1
WO2025226876A1 PCT/US2025/026068 US2025026068W WO2025226876A1 WO 2025226876 A1 WO2025226876 A1 WO 2025226876A1 US 2025026068 W US2025026068 W US 2025026068W WO 2025226876 A1 WO2025226876 A1 WO 2025226876A1
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WIPO (PCT)
Prior art keywords
protein
polynucleotide
target polynucleotides
sample
samples
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PCT/US2025/026068
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English (en)
Inventor
Martin Ranik
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Watchmaker Genomics Inc
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Watchmaker Genomics Inc
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Publication of WO2025226876A1 publication Critical patent/WO2025226876A1/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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein

Definitions

  • polynucleotide sequencing library normalization may be achieved by contacting a polynucleotide sequencing library with a predetermined amount of a ribonucleoprotein that binds to an adapter sequence of the polynucleotide library (e.g., a catalytically inactive CRISPR associated protein (dCAS)-guide RNA (gRNA) complex) and extracting the polynucleotides that (1) comprise the adapter and (2) are bound by the ribonucleoprotein complex.
  • adapter sequence may comprise an index which can be used for library normalization.
  • Adapter sequence indexes are used in multiplexed sequencing where multiple different samples are combined together and sequenced in the same experiment. Each sample is associated with an index with a sequence that can be used in bioinformatic analysis to determine which sample the polynucleotide came from. It is recognized herein that normalization in multiplexed sequencing libraries could be performed by targeting index regions of adapter. Additionally, the sequences of index regions are far less constrained than static regions of adapter sequences because static regions are used by the sequencer (e.g., for binding to an ILLUMINA sequencing flow cell) but index regions are not.
  • a Cas protein protospacer adjacent motif (PAM) or reverse complements of a PAM (rPAM) can easily be inserted into index regions (or adjacent to index regions) to allow for normalization using a guide RNA that is complementary to the index or a reverse complement of the index.
  • this disclosure provides a method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence of any one of SEQ ID NOs: 1-4;(ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the adapter sequence, or a reverse complement of the adapter sequence, of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas protein or a dArgonaute protein comprising an affinity tag, wherein the predetermined concentration of the dCas protein or the dArgonaute protein is cognate to the guide polynucleotide; (iii) contacting
  • the present disclosure provides a method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the index, or a reverse complement of the index, of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas protein or a dArgonaute protein comprising an affinity tag, wherein the predetermined concentration of the dCas protein or the dArgonaute protein is cognate to the guide polynucleotide; (iii) contacting the solution with a solid phase comprising an affinity tag
  • the targeting region is complementary to a static region of an adapter sequence or a reverse complement thereof.
  • the solutions produced from the at least two samples are combined into a single solution after step (ii) and before step (iii).
  • the present disclosure provides a method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising: (i) obtaining a mixture of the at least two samples, wherein the target polynucleotides of the at least two samples comprise an adapter sequence and an index, wherein the at least two samples do not have the same index, and, for each sample, obtaining a guide polynucleotide comprising a targeting region that is complementary to the index, or a reverse complement of the index, of the sample; (ii) producing a solution, the producing comprising combining (a) the mixture of the at least two samples, (b) a predetermined concentration of each a guide polynucleotide
  • the predetermined concentration of each guide polynucleotide is different. In some embodiments, the different predetermined guide polynucleotide concentrations normalize the target polynucleotides of the at least two samples in a predetermined stoichiometric ratio.
  • the present disclosure provides a method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index that is adjacent to a protospacer adjacent motif (PAM); (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a CRISPR-associated protein (Cas) guide RNA (gRNA) polynucleotide comprising a targeting region that is complementary to a reverse compliment of a portion of the adapter sequence that is adjacent to the 5’ end of the PAM of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas protein comprising an affinity tag, wherein the predetermined concentration of the dCas protein is cognate to the guide polyn
  • the solutions produced from the at least two samples are combined into a single solution after step (ii) and before step (iii).
  • the portion is 15-30 nucleotides.
  • the present disclosure provides a method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index that is adjacent to a reverse complement of a protospacer adjacent 12372134.1 motif (rPAM); (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a CRISPR-associated protein (Cas) guide RNA (gRNA) polynucleotide comprising a targeting region that is complementary to a portion of the adapter sequence that is adjacent to the 3’ end of the rPAM of
  • Cas CRISPR-associated
  • the solutions produced from the at least two samples are combined into a single solution after step (ii) and before step (iii).
  • the present disclosure provides a method of enriching a target polynucleotide in a sample, the method comprising: (i) obtaining the sample, wherein the target polynucleotide of the sample comprises an adapter sequence comprising an index; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that complementary to the index or a reverse complement of the index; and (c) a predetermined concentration of a dCas protein or a dArgonaute protein comprising an affinity tag, wherein the predetermined concentration of the dCas protein or the dArgonaute protein is cognate to the guide polynucleotide; (iii) contacting the solution with a solid phase comprising an affinity tag binding molecule that
  • the index region is adjacent to a PAM. In some embodiments, the PAM is adjacent to the 5’ terminal of the index. In some embodiments, the index region is adjacent to a rPAM. In some embodiments, the rPAM is adjacent to the 3’ terminal of the index. In some embodiments, the index comprises a nucleic acid sequence of any one of the index sequences described herein.
  • the guide polynucleotide comprises a CRISPR-associated protein (Cas) guide RNA (gRNA) polynucleotide or an Argonaute guide polynucleotide. In some embodiments, the Cas gRNA polynucleotide targeting region is a homology region.
  • the Cas gRNA polynucleotide comprises a homology region of any one of SEQ ID NOs: 5-10.
  • the Argonaute guide polynucleotide is an siRNA, miRNA, piRNA, shRNA or siDNA.
  • the guide polynucleotide is a DNA polynucleotide.
  • the guide polynucleotide is a RNA polynucleotide.
  • the guide polynucleotide comprises a modified nucleic acid.
  • the modified nucleic acid comprises 2′F RNA, 2′OMe RNA, and/or a phosphorothioate bonds (PS).
  • the guide polynucleotide is a Cas gRNA polynucleotide and the Cas gRNA polynucleotide does not comprise a modified nucleic acid in a position that interacts with a Cas protein cognate to the gRNA.
  • the dCas is a catalytically-inactive Cpf1, C2c1, C2c3, C2c2, CasX, or CasY protein.
  • the dCas protein comprises an amino acid sequence of SEQ ID NO: 13.
  • the dArgonaute is a catalytically-inactive LrAgo, PfAgo, TtAgo, AaAgo, AfAgo, MjAgo, MpAgo, NgAgo, RsAgo, CpAgo, IbAgo, KmAgo, or SeAgo.
  • the affinity tag binding molecule comprises Ni2+ and the affinity tag comprises a His Tag.
  • the affinity tag binding molecule comprises biotin and the affinity tag comprises avidin.
  • the affinity tag binding molecule comprises an anti-myc antibody and the affinity tag comprises a myc tag.
  • the affinity tag and corresponding affinity tag binding molecule are selected from Table 1.
  • the solid phase comprises magnetic beads.
  • separating the solution from the solid phase comprises immobilizing the solid phase and washing the solid phase.
  • extracting the target polynucleotides from the solid phase comprises combining the solid phase and protease.
  • extracting the target polynucleotides from the solid phase comprises combining the solid phase and proteinase K in a solution sufficient for proteinase K activity.
  • proteinase K digests the dCas protein or dArgonaute protein that is bound to the solid phase thereby extracting the target polynucleotides. 12372134.1
  • steps (i)-(v) are performed in sequential order.
  • normalizing comprises making the concentration of the of target polynucleotides between the at least two samples within 15% of one another after normalization.
  • the present disclosure provides a guide polynucleotide comprising a targeting region that is complementary to any one of SEQ ID NOs: 34, 22, 27, 29, or 1-4 or a reverse complement of any one of SEQ ID NOs: 34, 22, 27, 29, or 1-4.
  • the present disclosure provides a guide polynucleotide comprising a targeting region that is complementary to an index of any one of the index sequences described herein or a reverse complement the index sequences described herein.
  • the guide polynucleotide comprises a CRISPR-associated protein (Cas) guide RNA (gRNA) polynucleotide or an Argonaute guide polynucleotide.
  • Cas CRISPR-associated protein
  • gRNA guide RNA
  • the Cas gRNA polynucleotide targeting region is a homology region.
  • the Cas gRNA polynucleotide comprises a homology region of any one of SEQ ID NOs: 5-10.
  • the Argonaute guide polynucleotide is an siRNA, miRNA, piRNA, shRNA or siDNA.
  • the guide polynucleotide is a DNA polynucleotide.
  • the guide polynucleotide is an RNA polynucleotide. In some embodiments, the guide polynucleotide comprises a modified nucleic acid. In some embodiments, the modified nucleic acid comprises 2′F RNA, 2′Ome RNA, and/or a phosphorothioate bonds (PS). In some embodiments, the guide polynucleotide is a Cas gRNA polynucleotide and the Cas gRNA polynucleotide does not comprise a modified nucleic acid in a position that interacts with a Cas protein cognate to the gRNA.
  • PS phosphorothioate bonds
  • the present disclosure provides a kit comprising: (a) a guide polynucleotide as described herein; and (b) a Cas protein or an Argonaute protein, or a polynucleotide sequence encoding a Cas protein or an Argonaute protein.
  • the Cas protein is a catalytically-inactive Cas protein (dCas).
  • the Cas protein is a Cas9 protein that is catalytically-inactive (dCas9).
  • the Cas9 protein comprises a D10A mutation and a H840A mutation.
  • the Cas protein is a catalytically-inactive Cpf1, C2c1, C2c3, C2c2, CasX, or CasY protein.
  • the dCas protein comprises an amino acid sequence 12372134.1 having at least 95% identity to SEQ ID NO: 13.
  • the dCas protein comprises an amino acid sequence of SEQ ID NO: 13.
  • the Argonaute protein is catalytically-inactive (dArgonaute).
  • the Argonaute protein is a CbAgo protein that is catalytically-inactive.
  • the Argonaute protein is a LrAgo, PfAgo, TtAgo, AaAgo, AfAgo, MjAgo, MpAgo, NgAgo, RsAgo, CpAgo, IbAgo, KmAgo, or SeAgo protein that is catalytically- inactive.
  • the dArgonaute protein comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 15-21.
  • the dArgonaute protein comprises an amino acid sequence of any one of SEQ ID NOs: 15-21.
  • the kit further comprises a catalytically active Cas protein or a catalytically active Argonaute protein.
  • the catalytically active Cas protein comprises Cpf1, C2c1, C2c3, C2c2, CasX, Cas9, or CasY.
  • the catalytically active Argonaute protein comprises LrAgo, PfAgo, TtAgo, AaAgo, AfAgo, MjAgo, MpAgo, NgAgo, RsAgo, CpAgo, IbAgo, KmAgo, or SeAgo.
  • the guide polynucleotide is capable of binding to the Cas protein or the Argonaute protein to form a ribonucleoprotein complex and the ribonucleoprotein complex is capable of binding to an adapter sequence.
  • the kit further comprises a primer that is complementary to a portion of an adapter sequence.
  • the portion of the adapter sequence is located in the proximal portion of the adapter sequence.
  • the primer is not complementary to an adapter sequence that is complementary to the guide polynucleotide targeting region.
  • the present disclosure provides a reaction mixture comprising: (i) a plurality of target polynucleotides, wherein the target polynucleotides comprise an adapter sequence of any one of SEQ ID NOs: 34, 22, 27, 29, or 1-4; (ii) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the adapter sequence or a reverse complement of the adapter sequence; and (iii) a predetermined concentration of a dCas protein or a dArgonaute protein.
  • the present disclosure provides a reaction mixture comprising: (i) a plurality of target polynucleotides, wherein the target polynucleotides comprise an adapter sequence comprising an index; (ii) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the index or a reverse complement of the index; and (iii) a predetermined concentration of a dCas protein or a dArgonaute protein.
  • the present disclosure provides a reaction mixture comprising: (i) a plurality of target polynucleotides, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index that is adjacent to a PAM; (ii) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a reverse complement of a portion of the adapter sequence that is adjacent to the PAM of the target polynucleotides of the sample; and (iii) a predetermined concentration of a dCas protein or a dArgonaute protein.
  • the present disclosure provides a reaction mixture comprising: (i) a plurality of target polynucleotides, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index that is adjacent to a rPAM ;(ii) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a portion of the adapter sequence that is adjacent to the rPAM of the target polynucleotides of the sample; and (iii) a predetermined concentration of a dCas protein or a dArgonaute protein.
  • the predetermined concentration of the guide polynucleotide, or the dCas protein or the dArgonaute protein is lower than the concentration of target polynucleotide in the reaction mixture.
  • the dArgonaute protein comprises LrAgo, PfAgo, TtAgo, AaAgo, AfAgo, MjAgo, MpAgo, NgAgo, RsAgo, CpAgo, IbAgo, KmAgo, or SeAgo, or a catalytically inactive variant thereof, and the guide polynucleotides are cognate to the dArgonaute protein.
  • the dCas protein comprises catalytically inactive Cpf1, C2c1, C2c3, C2c2, CasX, Cas9, or CasY, or a variant thereof, and the guide polynucleotides are cognate to the dCas protein.
  • the reaction mixture further comprises a catalytically active Cas protein or a catalytically active Argonaute protein.
  • the present disclosure provides a ribonucleoprotein (RNP) complex comprising: (i) a guide polynucleotide provided herein; (ii) a Cas protein or an Argonaute protein that is cognate to the guide polynucleotide; and (iii) an adapter sequence of any one of SEQ ID NOs: 34, 22, 27, 29, or 1-4 or an adapter sequence comprising an index of any one of the index sequences described herein, wherein the targeting region of the guide polynucleotide is complementary to the adapter sequence and wherein the guide polynucleotide is cognate to the Cas protein or the Argonaute protein.
  • RNP ribonucleoprotein
  • the Argonaute protein comprises LrAgo, PfAgo, TtAgo, AaAgo, AfAgo, MjAgo, MpAgo, NgAgo, RsAgo, CpAgo, IbAgo, KmAgo, or SeAgo, or a catalytically 12372134.1 inactive variant thereof.
  • the Cas protein comprises Cpf1, C2c1, C2c3, C2c2, CasX, Cas9, or CasY, or a catalytically inactive variant thereof.
  • FIG.1 shows the distribution of ULTIMA Genomics libraries captured using dCas9 ribonucleoprotein (RNP) and ULTIMA Genomics single guide RNA (UG_sgRNA)1, UG_sgRNA_2, and UG_sgRNA_3.
  • FIGs. 2A-2D show a schematic of library molecule denaturation and partial sequence extension to produce a double stranded PAM site.
  • a library molecule is ligated with two Y-shaped adapters at the 3’ and 5’ ends.
  • the library molecule is denatured and bound to a partial primer at the 3’ end.
  • FIG. 1 shows the distribution of ULTIMA Genomics libraries captured using dCas9 ribonucleoprotein (RNP) and ULTIMA Genomics single guide RNA (UG_sgRNA)1, UG_sgRNA_2, and UG_sgRNA_3.
  • FIGs. 2A-2D show a schematic of library molecule denaturation and partial sequence extension to produce a double
  • the bound primer is extended with a polymerase, creating a complementary strand (dashed line), which does not span the entire 3’ adapter, but does comprise a double-stranded, full-length 5’ adapter, which contains a PAM site for dCas9 targeting.
  • FIG. 2D illustrates the binding of a dCas9 molecule to the double-stranded PAM site.
  • polynucleotide refers to polymers of nucleotides (e.g., deoxyribonucleotides, ribonucleotides and modified nucleotides).
  • the polymer may be in either single- or double- stranded form.
  • the term encompasses polynucleotides containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the naturally occurring nucleotides.
  • a “guide polynucleotide” refers a polynucleotide that (1) comprises a targeting region which is complementary to a target (e.g., an adapter sequence) and (2) that is capable of facilitating binding of a ribonucleoprotein complex comprising the guide polynucleotide to the target.
  • the guide polynucleotide is a CRISPR associated protein (Cas) guide RNA (gRNA) polynucleotide or an Argonaute guide polynucleotide.
  • a Cas gRNA polynucleotide refers to a two polynucleotide system comprising a tracrRNA and a crRNA e.g., as described in Karvelis T et al., RNA Biol. 2013 May;10(5):841- 51. PMID: 23535272.
  • the tracrRNA comprises a sequence encoding a stem loop structure that associates with the Cas protein (e.g., dCas9).
  • the crRNA comprises a homology region that is complementary to the target (e.g., an adapter sequence) and a region that is complementary to the tracrRNA.
  • the crRNA and the tracrRNA may form a complex with the Cas protein, which in turn binds to a target DNA or RNA polynucleotide (depending on the Cas type).
  • a Cas gRNA polynucleotide refers to a single guide RNA (sgRNA) polynucleotide e.g., as described in Jinek M et al., Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829.
  • a sgRNA comprises a sequence encoding a stem loop structure that associates with the Cas protein and a homology region.
  • Many Cas proteins bind to polynucleotides (e.g., double stranded DNA) in a position that is adjacent to a protospacer adjacent motif (PAM) site.
  • PAM protospacer adjacent motif
  • the reverse complement of the PAM is also called the rPAM.
  • the homology region is complementary to a portion of the adapter sequence that is adjacent to a PAM (e.g., sufficiently adjacent such that Cas binding to the adapter sequence can take place).
  • the homology region is complementary to a polynucleotide (e.g., an adapter of index) that is adjacent to the 5’ end of a PAM. In some embodiments, the homology region is complementary to a polynucleotide (e.g., an adapter of index) that is adjacent to the 3’ end of a rPAM.
  • Methods of making and using gRNAs are known in the art, e.g., as described in Mohr SE, et al., FEBS J. 2016 Sep;283(17):3232-8.
  • PMCID PMC5014588.
  • An Argonaute guide polynucleotide refers to a polynucleotide encoding a small interfering RNA (siRNA), microRNA (miRNA), P element-induced wimpy testis (PIWI)- interacting RNA (piRNA) and small interfering DNA (siDNA) e.g., as described in Wu J et al., J Adv Res. 2020 Apr 29;24:317-324, PMID: 32455006.
  • the Argonaute guide polynucleotide comprises a targeting region that is complementary to an adapter sequence as described herein.
  • a guide polynucleotide is single stranded (e.g., an 12372134.1 RNA Cas gRNA polynucleotide). In some embodiments, a guide polynucleotide is double stranded (e.g., double stranded DNA encoding a Cas gRNA polynucleotide, or dsRNA used an Argonaute guide polynucleotide). In some embodiments, a guide polynucleotide is an RNA molecule. In some embodiments, a guide polynucleotide is a DNA molecule.
  • the guide polynucleotide comprises modified nucleic acids (e.g., 2′F RNA, 2′OMe RNA, and/or a phosphorothioate bonds (PS)).
  • Nucleic acids in a guide polynucleotide e.g., an RNA guide polynucleotide like a Cas guide RNA
  • may be modified to increase the stability of the guide polynucleotide e.g., reduce nuclease digestion).
  • a Cas gRNA (e.g., a Cas9 gRNA) may comprise modified nucleic acids (e.g., 2′OMe) in regions of the gRNA that do not interact with the Cas protein (e.g., Cas9), and still maintain an ability to direct the Cas protein to the target, as described in Yin H et al., Nature Biotechnology 2017 Dec; 35(12): 1179-1187.
  • the Cas guide RNA polynucleotide comprises the modifications of the e-sgRNA as described in Yin et al. 2017.
  • a Cas9 sgRNA does not comprise a modified nucleic acid (e.g., a 2’OH modification) at one or more positions 22-27, 43-45, 47, 49, 51, 58, 59, 62, 63-65, 68-69, or 82 counted from the 5’ end of the sgRNA (i.e., with reference to an sgRNA of (GGGCGAGGAGCUGUUCACCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU, (SEQ ID NO: 11)).
  • a modified nucleic acid e.g., a 2’OH modification
  • the sgRNA comprises 2’-O-methyl RNA bases.
  • 2’-O-methyl RNA bases the 2’-hydroxyl (-OH) group of the ribose sugar in the RNA molecule is replaced by a methyl group (-CH3).
  • This modification affects the ribose sugar, and the nitrogenous bases (adenine, guanine, cytosine, uracil) themselves are not typically modified in this context.
  • the 2’-O-methyl RNA bases are 2’O-methyladenosine, 2’O-methylguanosine, 2’O-methylcytidine, and 2’O-methyluridine.
  • the sgRNA comprises the sequence: 5’- mA*mG*mA* rUrCrG rGrArA rGrArG rCrGrU rCrGrU rGrUrG rUrUrU rUrArG rArGrArGrUrArG rArGrUrUrArG rArArArGrUrU rArArGrUrU rArArArGrUrU rArArArGrUrCrArGrUrCrGrU rU rArU rCrArA rCrUrU rGrGrArUrU rCrArA rCrUrU rGrArArUrU rGrArArArArCrUrU rGrArArArG
  • a “targeting region” of a guide polynucleotide refers to a region of the guide polynucleotide that is complementary to a target polynucleotide sequence (e.g., an adapter sequence) or a reverse complement of the target polynucleotide sequence.
  • the targeting region comprises a portion that has the same sequence as the target polynucleotide sequence.
  • the guide polynucleotide is a Cas gRNA polynucleotide comprising a homology region.
  • a Cas gRNA polynucleotide homology region may comprise a series of contiguous amino acids that are complementary to a target polynucleotide sequence (e.g., an adapter sequence).
  • the homology region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length.
  • the homology region is 18-26 nucleotides in length.
  • the homology region is 17-24 nucleotides in length.
  • the homology region is 18-22 nucleotide in length.
  • the homology region is 20 nucleotides in length.
  • the homology region comprises a sequence that has the same sequence as a portion of the target polynucleotide sequence.
  • the guide polynucleotide is an Argonaute guide polynucleotide.
  • Argonaute guide polynucleotides may be RNA interference (RNAi) polynucleotides.
  • the Argonaute guide polynucleotide comprises a targeting region of a small interfering RNA (siRNA), microRNA (miRNA), P element-induced wimpy testis (PIWI)- interacting RNA (piRNA) and small interfering DNA (siDNA) e.g., as described in Wu J et al., J Adv Res.
  • the targeting region is complementary to an adapter sequence as described herein. In some embodiments, the targeting region is complementary to a static region (e.g., that generally does not change between adapters) of the adapter sequence. In some embodiments, the targeting region is complementary to a sequence comprising a region that was added to the sequence for the purpose of being complementary to a guide polynucleotide targeting region. For example, an additional sequence can be added to an adapter sequence for the purpose of being complementary to a guide polynucleotide targeting region (e.g., for use in library normalization).
  • Such an additional sequence may be a sequence that is specifically bound by a Cas protein-gRNA complex or an Argonaute guide polynucleotide complex compared to other sequences in a polynucleotide sequencing library.
  • the targeting region is complementary to a next-generation sequencing adapter sequence (e.g., an ELEMENT sequence adapter, a PACBIO sequence 12372134.1 adapter, or an ULTIMA sequence adapter).
  • ELEMENT, PACBIO, and ULTIMA sequence adapters are known in the art and exemplary adapters are in Table 2.
  • the targeting region is complementary to an index within an adapter sequence.
  • index refers a polynucleotide within an adapter than can be used to identify the sample from which the polynucleotide came. For example, 5’ AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC[index]ATCTCGTATGCCGTCTTC TGCTTG 3‘ (SEQ ID NO: 22).
  • Adapters with indexes are typically used when combining multiple different samples together prior to sequencing .For example, prior to combining polynucleotide from different samples, the polynucleotides of the different samples are modified to comprise adapter sequences with an index. Polynucleotides of each sample have a different index. The different samples are then combined and sequenced.
  • the index is 2-40 nucleotides (SEQ ID NO: 22 and SEQ ID NOs: 529-566). In some embodiments, the index is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the index is 20, 25, 30, 35, or 40 or more nucleotides. In some embodiments, the nucleotides of the index may be any combination or permutation of nucleotides A, T, G, or C. In some embodiments, index sequences do not comprise restriction cut sites that are used in preparations for sequencing. Index sequences are additionally known in the art.
  • ILLUMINA adapters frequently have index sequences as described in ILLUMINA document #1000000002694v16 (thesequencingcenter.com/wp-content/uploads/2021/12/illumina-adapter-sequences.pdf accessed April 19, 2024), the index sequences of which are incorporated by reference in their entirety.
  • a targeting region is complementary to an index region.
  • a targeting region that is complementary to an index may also refer to targeting region that is complementary to a reverse complement of the index.
  • the guide polynucleotide may also be complementary to nucleotides extending from the 5’ terminal and/or 3’ terminal of the index within the adapter sequence.
  • the index region is GTTATGCT and the adapter region comprising the index is ...GGCGGGTTATGCTATTTCCT (SEQ ID NO: 23)
  • the targeting region may be complementary to the index region and the nucleotide 3’ and/or 5’ of the index.
  • the targeting region is complementary to the reverse complement of the index region and the nucleotide 3’ and/or 5’ of the index.
  • the adapter sequence is modified to comprise a Cas protein protospacer-adjacent motif (PAM) or a reverse complement of a PAM (rPAM)).
  • a “protospacer-adjacent motif (PAM)” is a nucleotide motif (often 3 consecutive nucleotides in the polynucleotide) that is required for Cas protein binding to the target polynucleotide.
  • the PAM is typically located on the DNA strand that is not complementary to the homology region of the guide RNA. In some embodiments, the PAM is adjacent to the 3’ end of the homology region of a Cas9 guide RNA.
  • the canonical Cas9 PAM sequence is 5’-NGG, wherein N is any nucleotide A, G, C, or T.
  • the adapter sequence is modified to comprise a PAM or rPAM to facilitate binding of the Cas-gRNA complex to a double stranded version of the target polynucleotide.
  • a PAM sequence or a rPAM sequence may be added to the 3’ end or 5’ end of the adapter sequence or index.
  • the PAM or rPAM is adjacent to the index in the adapter sequence.
  • “Adjacent” as used in the context of two different polynucleotides means the two different polynucleotides are covalently bound to another with no intervening nucleotides between them. For example, if a PAM sequence is CGG and an index sequence is TTATCGTG the PAM and index would be adjacent in one of two configurations: CGGTTATCGTG (SEQ ID NO: 24) (PAM is on the 5’ terminal of the index) or TTATCGTGCGG (SEQ ID NO: 25) (PAM is on the 3’ terminal of the index).
  • the PAM and index would not be adjacent to another if there were an intervening nucleic acid between them in the primary sequence, e.g., TTATCGTGACGG (SEQ ID NO: 26).
  • the PAM or rPAM is on the 5’ terminal of the index. In some embodiments, the PAM or rPAM is on the 3’ terminal of the index. In some embodiments, the PAM or rPAM is in the index. In some embodiments, the PAM or rPAM is adjacent to a reverse complement of an index. In some embodiments, the PAM or rPAM is adjacent to the 5’ end of a reverse complement of an index.
  • the PAM or rPAM is adjacent to the 3’ end of a reverse complement of an index.
  • complementary refers to the degree of expected Watson-Crick base pairing between a first polynucleotide (e.g., a guide polynucleotide targeting region) and a second polynucleotide (e.g., an adapter sequence).
  • Complementary nucleotides are, generally, A and T (or A and U), and G and C.
  • complementary refers to 100% 12372134.1 complementarity between two polynucleotides (e.g., a guide polynucleotide targeting region and an adapter sequence).
  • complementary refers to 70%, 80%, 90%, 95%, or 99% complementarity between two polynucleotides.
  • a targeting region may be complementary to an adapter sequence when one, two, or three nucleic acids in the targeting region are not complementary to the adapter sequence.
  • complementary refers to a sufficiently degree of Watson-Crick base pairing between a guide polynucleotide targeting region (of a RNP (e.g., dCas9 bound to a guide RNA) and an adapter sequence for the RNP to bind to the adapter sequence.
  • complementary refers to a sufficient degree of Watson-Crick base pairing between an Argonaute guide polynucleotide (e.g., a miRNA, siRNA, pwRNA, shRNA, or siDNA) and a target sequence (e.g., an adapter sequence) to direct binding of an Argonaute protein comprising the Argonaute guide polynucleotide to bind to the target.
  • a target sequence e.g., an adapter sequence
  • An “adapter sequence” refers to a polynucleotide added (e.g., ligated) to an end (e.g., 3’ and/or 5’) of a target polynucleotide for use in sequencing of the polynucleotide.
  • the adapter sequence refers to a sequence of nucleic acids encoding the adapter.
  • the adapter sequence may be used for sequencing using a particular sequencing platform.
  • ILLUMINA i5/p5 and i7/p7 adapter sequences may be used for sequencing using the ILLUMINA sequencing platform or ULTIMA Genomics ppmSeq adapter sequences may be used for sequencing using an ULTIMA Genomics sequencing platform.
  • the adapter sequence comprises a static region (e.g., that generally does not change between adapters) and a dynamic region (e.g., that may change between adapters).
  • the adapter sequence comprises a distal region (generally static), an index region (generally dynamic), and a proximal region (generally static).
  • an ILLUMINA adapter sequence may comprise a p5 region which is static, an index region which is dynamic, and an i5 region which is static; or an ULTIMA Genomics adapter sequence may comprise a ppmSeq adapter sequence, an index region which is dynamic, and a region which is static.
  • the guide polynucleotide targeting region is complementary to a static region of the adapter sequence. In some embodiments, the guide polynucleotide targeting region is complementary to the dynamic region of the adapter sequence.
  • the guide polynucleotide comprises a targeting region that is complementary to a static region of an adapter sequence (e.g., a distal region or a proximal region of an adapter sequence).
  • the guide polynucleotide comprises a targeting region that is complementary to a dynamic region of an adapter sequence (e.g., an index).
  • at least some of the target polynucleotides comprise the same static adapter sequences (e.g., the same distal and/or proximal region).
  • the majority of the target polynucleotides comprise the same static adapter sequence.
  • the adapter sequence comprises an index.
  • the adapter sequence is an adapter sequence used in ILLUMINA, ION TORRENT, PACBIO, ELEMENT, ULTIMA, OMNIOME, SINGULAR, or MGI sequencing.
  • the adapter sequence used is a PACBIO, ELEMENT, or ULTIMA adapter sequence.
  • an adapter sequence is a PACBIO adapter sequence.
  • an adapter sequence is an ELEMENT adapter sequence.
  • an adapter sequence is an ULTIMA adapter sequence.
  • the adapter sequence is an adapter sequence from any one of the following kits: a WATCHMAKER DNA Library Prep Kit with Fragmentation or a WATCHMAKER RNA Library Prep Kit with Polaris Depletion ILLUMINA TruSeq PCR-Free Library Preparation Kit, a TruSeq Nano DNA Library Prep Kit, a NEXTERA DNA Library Prep Kit, a NEXTERA DNA XT Library Prep Kit, a NEXTERA Rapid Capture Exome Kit, a NEXTERA Rapid Capture Expanded Exome Kit, an AmpliSeq for ILLUMINA Library Prep Kit, an ILLUMINA RNA Prep with Enrichment Kit, an ILLUMINA Stranded mRNA Prep Kit, a TruSeq RNA Library Prep Kit, a TruSeq Stranded Total RNA Kit, a TruSeq Stranded mRNA Kit, a TruSeq Small RNA Kit, or an ULTIMA pp
  • the adapter sequence is an adapter sequence used in any one of the following kits: a NEBNEXT Fast DNA for ION TORRENT, a NEBNEXT Fast DNA Fragmentation & Library Prep Set for ION TORRENT, a THERMOFISHER Precision ID Library Kit, a THERMOFISHER Ion Xpress Plus Fragment Library Kit, a THERMOFISHER Ion Xpress Barcode Adapters 1-96 Kit, and/or a THERMOFISHER Ion AmpliSeq Transcriptome Human Gene Expression Kit.
  • the adapter sequence is an adapter sequence used in any one of the following kits: a PACBIO SMRTbell Template Prep Kit 1.0, a SMRTbell Barcoded Adapter Complete Prep Kit-96, or a Barcoded Adapter Kit.
  • the targeting region may be complementary to an adapter sequence used in a QIAGEN QIAseq Stranded RNA Library Kit or a QIAseq UPX 3’ Transcriptome Kit, a Perkin Elmer NEXTFLEX Rapid Directional RNA- 12372134.1 Seq Kit or a NEXTFLEX Small RNA-Seq Kit, or a Takara Bio SMART-Seq mRNA Kit or a SMART-Seq mRNA LP Kit.
  • the adapter sequence comprises a shared Y-adapter sequence.
  • the shared Y-adapter sequence is a 13 bp sequence.
  • the shared Y-adapter sequence is a 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp sequence.
  • Kits In some aspects, this disclosure describes a kit comprising: (a) a guide polynucleotide described herein; and (b) a Cas protein or an Argonaute protein, or a polynucleotide sequence encoding a Cas protein or an Argonaute protein (e.g., as described herein). In some embodiments, the kit comprises a Cas protein.
  • a “Cas protein” refers to a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the Cas protein has nuclease activity. In some embodiments, the Cas protein does not have nuclease activity (dCas). In some embodiments, the Cas protein has nicking activity (nickase). In some embodiments, the Cas protein is a Cas9 protein. “Cas9” refers to a Cas9 protein or a fragment thereof present in any bacterial species that encodes a Type II CRISPR/Cas9 system. See, for example, Makarova et al., Nature Reviews, Microbiology, 9: 467-477 (2011), including supplemental information.
  • Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae.
  • An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi et al., 2013, RNA Biol.
  • Full-length Cas9 is an RNA-mediated endonuclease comprising a recognition domain and two nuclease domains (HNH and RuvC, respectively).
  • HNH is linearly continuous
  • RuvC is separated into three regions, one left of the recognition domain, and the other two right of the recognition domain flanking the HNH domain.
  • Cas9 from Streptococcus pyogenes is targeted to a genomic site in a 12372134.1 cell by interacting with a guide RNA that hybridizes to a 20-nucleotide DNA sequence that immediately precedes an NGG motif recognized by Cas9.
  • the Cas9 is a catalytically-inactive or nuclease-dead Cas9 (dCas9) that has been modified to inactivate Cas9 nuclease activity. Modifications include, but are not limited to, altering one or more amino acids to inactivate the nuclease activity or the nuclease domain.
  • D10A, H840A, and/or R1335K mutations can be made in Cas9 from Streptococcus pyogenes to inactivate Cas9 nuclease activity.
  • the Cas9 protein comprises a D10A mutation and a H840A mutation.
  • Other modifications include removing all or a portion of the nuclease domain of Cas9, such that the sequences exhibiting nuclease activity are absent from Cas9.
  • a catalytically- inactive Cas9 may include polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity.
  • the catalytically-inactive Cas9 retains the ability to bind to DNA even though the nuclease activity has been inactivated. Accordingly, dCas9 includes the polypeptide sequence or sequences required for DNA binding but includes modified nuclease sequences or lacks nuclease sequences responsible for nuclease activity. In some embodiments, the catalytically-inactive Cas9 protein is a full-length Cas9 sequence from S. pyogenes lacking the polypeptide sequence of the RuvC nuclease domain and/or the HNH nuclease domain and retaining the DNA binding function.
  • the catalytically-inactive Cas9 protein sequences have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to Cas9 polypeptide sequences lacking the RuvC nuclease domain and/or the HNH nuclease domain and retains DNA binding function.
  • the Cas protein is Alt-RTM S.p. dCas9 Protein V3 (IDT).
  • the Cas protein is a Cpf1 (Cas12a), C2c1, C2c3, C2c2, CasX, or CasY protein.
  • the Cas protein has been modified to inactivate Cas9 nuclease activity.
  • Modifications include, but are not limited to, altering one or more amino acids to inactivate the nuclease activity or the nuclease domain of the Cas protein.
  • D908, D832, E993, R1226, and/or D1235 mutations can be made in Cpf1 from Acidaminacoccus sp. BV3L6, Lachnospiraceae ND2006, or Francisella tularensis subsp. novicida U112, to inactivate Cpf1 nuclease activity.
  • Other modifications include removing all or a portion of the nuclease domain, such that the sequences exhibiting nuclease activity are absent.
  • a catalytically-inactive Cas protein may include polypeptide sequences 12372134.1 modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity.
  • the catalytically-inactive Cas protein retains the ability to bind to DNA even though the nuclease activity has been inactivated.
  • catalytically-inactive Cas protein includes the polypeptide sequence or sequences required for DNA binding but includes modified nuclease sequences or lacks nuclease sequences responsible for nuclease activity.
  • the catalytically-inactive Cas protein is a full-length Cas sequence lacking the polypeptide sequence of the nuclease domain and retaining the DNA binding function. In other embodiments, the catalytically-inactive Cas protein sequences have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to Cas polypeptide sequences lacking the nuclease domain and retains DNA binding function. In some embodiments, the Cas protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 13 or SEQ ID NO: 14. In some embodiments, the Cas protein comprises an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the dCas protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 13. In some embodiments, the dCas protein comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the Cas protein comprise a nuclease localization sequence. In some embodiments, the kit comprising an Argonaute protein.
  • An “Argonaute protein” refers to a protein that binds small noncoding nucleic acids (e.g., an Argonaute guide polynucleotide) and utilizes them for the guided cleavage of complementary nucleic acid targets or indirect gene silencing by recruiting additional factors.
  • Catalytically-active Argonaute proteins are capable of nucleic acid guided binding to a complementary nucleic acid target (e.g., DNA) and cleavage of the nucleic acid target. See, e.g., Kaya et al., 2016, PNAS 113(5):4057- 62.
  • the prokaryotic Argonaute (AGO) gene family encodes several domains: N-terminal (N), PAZ, MID, and a C-terminal PIWI domain.
  • the MID and PAZ domains are responsible for binding of the 5’-end and 3’-end, respectively, of a guide polynucleotide.
  • the Ago PIWI domain comprises nuclease activity.
  • the Argonaute protein is catalytically-inactive.
  • the Argonaute protein is a CbAgo protein that is catalytically-inactive.
  • the Argonaute protein is a LrAgo, PfAgo, TtAgo, AaAgo, AfAgo, MjAgo, MpAgo, NgAgo, RsAgo, CpAgo, IbAgo, KmAgo, or SeAgo protein that is catalytically- 12372134.1 inactive.
  • the Argonaute protein has been modified to inactivate nuclease activity or inherently has reduced nuclease activity (see, e.g., Kaya et al., 2016, PNAS 113(5):4057-62).
  • Modifications may include, but are not limited to, altering one or more amino acids to inactivate the nuclease activity or the nuclease domain of the Argonaute protein.
  • the catalytically-inactive Argonaute protein is a CbAgo protein that comprises a D541A mutation and a D611A mutation. See, e.g., Hegge et al., 2019, Nuc. Acids Res. 47(11):5809-21.
  • all or a portion of the nuclease domain may be removed, such that the sequences exhibiting nuclease activity are absent.
  • a catalytically-inactive Argonaute protein may include polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity.
  • the catalytically-inactive Argonaute protein retains the ability to bind to DNA even though the nuclease activity has been inactivated.
  • catalytically-inactive Argonaute protein includes the polypeptide sequence or sequences required for DNA binding but includes modified nuclease sequences or lacks nuclease sequences responsible for nuclease activity.
  • the catalytically-inactive Argonaute protein is a full-length Argonaute sequence lacking the polypeptide sequence of the nuclease domain and retaining the DNA binding function. In other embodiments, the catalytically-inactive Argonaute protein sequences have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to the Argonaute polypeptide sequences lacking the nuclease domain and retains DNA binding function.
  • the dArgonaute protein comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 15-21. In some embodiments, the dArgonaute protein comprises an amino acid sequence of any one of SEQ ID NOs: 15-21.
  • the kit comprises a catalytically inactive Cas protein (dCas protein) or a catalytically inactive Argonaute protein (e.g., dArgonaute) as described herein.
  • the kit comprises a catalytically inactive Cas protein (dCas protein) and a catalytically active Cas protein as described herein.
  • the kit comprises a catalytically a catalytically inactive Argonaute protein (e.g., dArgonaute) a catalytically active Argonaute protein as described herein.
  • the kit comprises a dCas and a cognate dCas guide RNA polynucleotide. In some embodiments, the kit comprises a dCas9 and a cognate dCas9 guide RNA. In some embodiments, the kit comprises a dArgonaute and a cognate dArgonaute guide polynucleotide. 12372134.1 In some embodiments, the kit comprises a Cas protein comprising an affinity tag or a Argonaute protein comprising an affinity tag.
  • the affinity tag is Albumin-binding protein (ABP), Alkaline Phosphatase (AP), AU1 epitope, AU5 epitope, Bacteriophage T7 epitope (T7-tag), Bacteriophage V5 epitope (V5-tag), Biotin-carboxy carrier protein (BCCP), Bluetongue virus tag (B-tag), Calmodulin binding peptide (CBP), Chloramphenicol Acetyl Transferase (CAT), Cellulose binding domain (CBP), Chitin binding domain (CBD), Choline-binding domain (CBD), Dihydrofolate reductase (DHFR), E2 epitope, FLAG epitope, Galactose-binding protein (GBP), Green fluorescent protein (GFP), Glu-Glu (EE-tag), Glu-Glu (EE-tag), Human influenza hemagglutinin (HA), HaloTag®, Histidine affinity tag (HAT), Horseradish Peroxidase (ABP), Al
  • the affinity tag and corresponding affinity tag binding molecule in a kit or for use in the method, are selected from Table 1.
  • Table 1 Affinity tag and affinity tag binding molecule.
  • the kit comprises a guide polynucleotide that is capable of binding to the Cas protein (e.g., cognate the guide polynucleotide is cognate to the Cas protein) or the Argonaute protein of the kit to form a ribonucleoprotein complex and the ribonucleoprotein complex is capable of binding to an adapter sequence.
  • cognate refers to a guide polynucleotide that is compatible with the Cas protein or the Argonaute protein (e.g., the guide polynucleotide is capable of directing the Cas protein or the Argonaute protein to a target polynucleotide).
  • a Cas9 sgRNA is cognate to Cas9 and dCas9.
  • binds or “specifically binds,” and like terms, refer to a molecule (e.g., a Cas- gRNA complex or a Argonaute-guide polynucleotide complex) that binds to a target nucleic acid with at least 2-fold greater affinity than non-target nucleic acids, e.g., at least any of 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for the target nucleic acid compared to an unrelated nucleic acid when assayed under the same binding affinity assay conditions.
  • a target nucleic acid with at least 2-fold greater affinity than non-target nucleic acids, e.g., at least any of 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50
  • the term “binds” or “specifically binds,” as used herein, can be exhibited, for example, by a molecule (e.g., a Cas-gRNA complex or a Argonaute-guide polynucleotide complex) having an equilibrium dissociation constant KD for the target nucleic acid of, e.g., 10 -2 M or smaller, e.g., 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M.
  • an antibody has a KD of less than 10 nM or less than 100 nM.
  • the kit further comprises a primer that is complementary to a portion of an adapter sequence (e.g., as described herein).
  • the primer is complementary to a proximal portion of an adapter sequence.
  • This primer may be used for creating double stranded target nucleotides comprising a double stranded proto-spacer adjacent motif (PAM) site that a dCas protein (e.g., as described herein) can use for binding.
  • PAM double stranded proto-spacer adjacent motif
  • the primer binds to a proximal portion of the adapter sequences such that the new polynucleotide (which is complementary to the target polynucleotide) does not comprise the distal portion of the adapter sequence and thus cannot be sequenced using next-generation sequencing (e.g., ILLUMINA Sequencing, ULTIMA Genomics Sequencing).
  • next-generation sequencing e.g., ILLUMINA Sequencing, ULTIMA Genomics Sequencing.
  • this method may be advantageous compared to PCR based methods because errors (e.g., mutations) introduced during elongation of the primer are not sequenced, but double stranded target polynucleotides may still be produced and bound be dCas proteins.
  • the primer is a DNA primer.
  • the DNA primer comprises nucleic acid modifications (e.g., as described herein).
  • the primer is complementary to a 3’ adapter sequence.
  • the primer is complementary to a 5’ adapter sequence.
  • the primer is not complementary to an adapter sequence that is complementary to the guide polynucleotide targeting region.
  • the primer when bound to the adapter sequence and elongated does not elongate the entire adapter sequence.
  • this disclosure describes a reaction mixture comprising: (i) a plurality of target polynucleotides, wherein the target polynucleotides comprise an ELEMENT, a PACBIO, or an ULTIMA adapter sequence; (ii) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the adapter sequence; and (iii) a predetermined concentration of a dCas protein or a dArgonaute protein.
  • the reaction mixture comprises an aqueous solution (e.g., a buffer that is suitable for performing a method described herein).
  • the reaction mixture comprises a predetermined concentration of dCas9 protein). In some embodiments, the reaction mixture comprises a predetermined concentration of dArgonaute protein. In some embodiments, the reaction mixture comprises target polynucleotides comprising an adapter sequence of any one of SEQ ID NOs: 1-4, 22, 27, 29, or 34. In some embodiments, the reaction mixture comprises a predetermined concentration of a guide polynucleotide of any one of SEQ ID NOs: 5-10.
  • this disclosure provides a reaction mixture comprising: (i) a plurality of target polynucleotides, wherein the target polynucleotides comprise an adapter sequence comprising an index; (ii) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the index; and (iii) a predetermined 12372134.1 concentration of a dCas protein or a dArgonaute protein.
  • the index comprises a nucleic acid sequence of any one of the index sequences described herein.
  • the adapter sequence comprises a PAM or rPAM adjacent to the index.
  • the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 50 femtomoles (fmols) to 3,400 fmols. In some embodiments, the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 200 fmols to 3,400 fmols. In some embodiments, the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 300 fmols to 3,400 fmols.
  • the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 500 fmols to 3,400 fmols. In some embodiments, the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 750 fmols to 3,400 fmols. In some embodiments, the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 1,000 fmols to 3,400 fmols. In some embodiments, the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 1,000 fmols to 3,400 fmols.
  • the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 2,000 fmols to 3,400 fmols. In some embodiments, the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 50 fmols. In some embodiments, the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 3,400 fmols. In some embodiments, the reaction mixture comprises a plurality of target polynucleotides comprising an adapter sequence at a concentration of 500 fmols to 3,400 fmols.
  • the reaction mixture comprises a predetermined concentration of dCas9 of SEQ ID NO: 13, target polynucleotides comprising an adapter sequence of any one of SEQ ID NO: 1-4, 22, 27, 29, or 34, and a predetermined concentration of a guide polynucleotide (e.g., an RNA guide polynucleotide) that comprises a homology region that is complementary to the adapter sequence.
  • a guide polynucleotide e.g., an RNA guide polynucleotide
  • Ribonucleoprotein (RNP) complexes 12372134.1 in some aspects this disclosure provides a ribonucleoprotein (RNP) complex comprising: (i) a guide polynucleotide; (ii) a Cas protein (e.g., dCas) or an Argonaute protein (e.g., dArgonaute); and (iii) an ELEMENT adapter sequence, a PACBIO adapter sequence, or an ULTIMA adapter sequence; wherein the targeting region of the guide polynucleotide is complementary to the adapter sequence.
  • a guide polynucleotide comprising: (i) a guide polynucleotide; (ii) a Cas protein (e.g., dCas) or an Argonaute protein (e.g., dArgonaute); and (iii) an ELEMENT adapter sequence, a PACBIO adapter sequence, or an ULTIMA adapter sequence
  • the RNP complex comprises (i) a RNA Cas gRNA polynucleotide comprising a homology region of any one of SEQ ID NOs: 5-10 (ii) a dCas protein of SEQ ID NO: 37, and (iii) an adapter sequence of any one of SEQ ID NOs: 1-4, 22, 27, 29, or 34; wherein the homology region of the RNA Cas gRNA polynucleotide is complementary to the adapter sequence.
  • the RNP complex comprises (i) a RNA Cas gRNA polynucleotide (ii) a Cas protein as described herein, and (iii) an adapter sequence comprising an index; wherein the homology region of the RNA Cas gRNA polynucleotide is complementary to the index.
  • the adapter sequence is unmodified.
  • the adapter sequence is unmodified at the 5’ end.
  • the RNP complexes are present at a concentration of 250 fmols to 14,000 fmols. In some embodiments, the RNP complexes are present at a concentration of 300 fmols to 14,000 fmols.
  • the RNP complexes are present at a concentration of 2,000 fmols to 14,000 fmols. In some embodiments, the RNP complexes are present at a concentration of 6,000 fmols to 14,000 fmols. In some embodiments, the RNP complexes are present at a concentration of 10,000 fmols to 14,000 fmols. In some embodiments, the RNP complexes are present at a concentration of at least 250 fmols . In some embodiments, the RNP complexes are present at a concentration of at least 300 fmols. In some embodiments, the RNP complexes are present at a concentration of at least 2,000 fmols.
  • the RNP complexes are present at a concentration of at least 6,000 fmols. In some embodiments, the RNP complexes are present at a concentration of at least 10,000 fmols. In some embodiments, the RNP complexes are present at a concentration of 250 fmols. In some embodiments, the RNP complexes are present at a concentration of 300 fmols. In some embodiments, the RNP complexes are present at a concentration of 2,000 fmols. In some embodiments, the RNP complexes are present at a concentration of 6,000 fmols. In some embodiments, the RNP complexes are present at a concentration of 10,000 fmols.
  • the RNP complexes are present at a concentration of 14,000 fmols.
  • Table 2 Sequences 12372134.1 12372134.1 12372134.1 Index Sequences Table 3: ILLUMINA TruSeq Indexes: 12372134.1 12372134.1 12372134.1 12372134.1 12372134.1 12372134.1 12372134.1 Table 4: IDT Xgen UDI/UMI Index sequences: 12372134.1 12372134.1 12372134.1 12372134.1 Table 5: Additional Index Sequences (can be used on i5 or i7 side): 1 CCATGTCAGT 2 ATTGATGCCG 3 GCCATTAGGA 4 ACATCCACTC 5 GATGTAGCGA 6 GCTGTTCCAA 7 TCTCCTGGAT 8 GATCCTGACA 9 TGCGATTACG 10 TGATAGGCTC 11 TAAGGCCTTC 12 CCTTAAGGTC 13 AGCTAACGGA 14
  • Normalizing refers to the process of producing a subsequent sample with a desired concentration of target polynucleotides from an initial sample having a different starting concentration of target polynucleotides than the subsequent sample.
  • “normalization” of two or more samples results in the production of two or more subsequent samples each having a concentration of target polynucleotides that are more similar to each other after performing the normalization methods than before performing the normalization methods.
  • a first sample e.g., a first polynucleotide library
  • a second sample e.g., a second polynucleotide library
  • the difference in concentration between the first sample and the second sample would be less than 5-fold (e.g., less than 4-fold, less than 3-fold, or less than 2-fold).
  • normalization of two or more samples results in the production of two or more subsequent samples each having equimolar (i.e., 1:1) concentrations.
  • the method of normalization results in differences in target polynucleotide concentration between two or more samples being less than 50% (e.g., less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 2.5%).
  • the method of normalization results in differences in target polynucleotide concentration between two or more samples being 5%-40%, 5%-30%, 5%-20%, 5%-10%, 10%-40%, 10%-30%, or 10%-20% in concentration.
  • the difference in starting target polynucleotide concentration between two or more samples may be relatively small (e.g., less than 5-10%) prior to performing a method of normalization described herein.
  • the method of normalization may be performed but the two or more samples may not have a detectably more similar concentration after normalization than before normalization.
  • normalization refers to altering the concentration of polynucleotide in at least two samples to achieve a given stoichiometric ratio of polynucleotides between the at least two samples. For example, a desired stoichiometric ratio for polynucleotides in a first sample and polynucleotides in a second sample may be 5:1.
  • normalization comprises multiplexing multiple samples to normalize the concentration of target polynucleotides in the multiple samples. “Multiplexing” refers to the processing of a single pooled sample made by combining multiple samples into the single pooled sample.
  • multiplexing therefore eliminates the need to quantify the amount of polynucleotides in the multiple samples and to subsequently adjust the volume of the samples prior to pooling or combine different volume of the samples (based on concentration) when pooling.
  • multiplexing may be accomplished by contacting each sample with a predetermined amount of a ribonucleoprotein (RNP) that binds to the target polynucleotides, pooling the samples in a multiplex normalization reaction, and extracting target polynucleotide that are bound by the RNP.
  • RNP ribonucleoprotein
  • the predetermined amount of RNP e.g., the moles of RNP added
  • the predetermined amount of RNP determines the molar amount of target polynucleotides pooled from that sample (so long as the predetermined amount of RNP is limiting compared the target polynucleotides).
  • This pooling of the libraries allows the target polynucleotides to be eluted at a desired molar concentration without needing to determine the concentrations of target polynucleotides in the individual samples, and without having to adjust the volume of the 12372134.1 samples prior to pooling or combine different volume of the samples (based on concentration) when pooling.
  • Target polynucleotide refers to a polynucleotide that comprises nucleic acids encoding an adapter sequence (e.g., an adapter sequence described herein) comprising an index, an ELEMENT adapter sequence, a PACBIO adapter sequence, or an ULTIMA adapter sequence (e.g., SEQ ID NOs: 1-4, 22, 27, 29, and 34) or a static region of an ELEMENT, PACBIO, or ULTIMA sequence.
  • an adapter sequence e.g., an adapter sequence described herein
  • an ELEMENT adapter sequence e.g., an ELEMENT adapter sequence described herein
  • a PACBIO adapter sequence e.g., an ELEMENT adapter sequence described herein
  • ULTIMA adapter sequence e.g., SEQ ID NOs: 1-4, 22, 27, 29, and 34
  • the target polynucleotide comprises a polynucleotide that comprises (1) adapter sequence comprising an index, an ELEMENT, PACBIO, or ULTIMA adapter sequence (e.g., SEQ ID NOs: 1-4, 22, 27, 29, or 34) or a static region of an ELEMENT, PACBIO, or ULTIMA sequence and (2) nucleic acids encoding a sequence of interest.
  • the target polynucleotide comprises an adapter sequence comprising a PAM adjacent to an index.
  • the target polynucleotide comprises nucleic acids encoding an adapter sequence described herein and nucleic acids encoding a sequence of interest.
  • the sequence of interest may be any sequence for which normalization is desired.
  • a sequence of interest may comprise, but is not limited to, DNA, genomic DNA, circulating tumor DNA, RNA, rRNA, mRNA, miRNA, or cDNA.
  • the adapter sequence may be ligated to the 5’ end and/or 3’ end of the sequence of interest.
  • the target polynucleotide comprises a first adapter sequence (e.g., SEQ ID NO: 1) on the 5’ end of the sequence of interest and a second adapter sequence (e.g., SEQ ID NO: 2) on the 3’ end of target sequence.
  • the target polynucleotide is double stranded DNA.
  • sample refers to a composition or solution comprising one or more target polynucleotides.
  • the sample comprises a plurality of target polynucleotides.
  • a “plurality” refers to two or more.
  • a plurality of target polynucleotide comprises two or more target polynucleotides.
  • the plurality of target polynucleotides comprises multiple copies of a single target polynucleotide sequence (e.g., at least 10, at least 100, at least 1000, at least 10,000, at least 100,000, or at least 1,000,000, or at least 10,000,000, at least 100,000,000 target polynucleotides).
  • the plurality of target polynucleotides comprises different target polynucleotide sequences (e.g., at least 10, at least 100, at least 1000, at least 10,000, at least 100,000, or at least 1,000,000, or at least 10,000,000, at least 100,000,000 different target polynucleotides), but at least some of the target polynucleotides (e.g., all of the target polynucleotides) comprise the 12372134.1 same adapter sequence.
  • the sample comprises a target polynucleotide and other molecules (e.g., polynucleotides that are not target polynucleotides and do not comprise an adapter sequence or comprise a different adapter sequence from the one complementary to the guide polynucleotide).
  • the sample comprises target polynucleotides comprising nucleic acids encoding genomic DNA.
  • the sample comprises target polynucleotides comprising nucleic acids encoding an RNA.
  • the sample comprises target polynucleotides comprising nucleic acids encoding a cDNA (e.g., of mRNA or lncRNA).
  • the sample comprises target polynucleotides comprising nucleic acids encoding an exon. In some embodiments, the sample comprises target polynucleotides comprising nucleic acids encoding an intron. In some embodiments, the plurality of target polynucleotides in the sample is present at a concentration of 50 fmols to 3,400 fmols. In some embodiments, the plurality of target polynucleotides is present at a concentration of 200 fmols to 3,400 fmols. In some embodiments, the plurality of target polynucleotides is present at a concentration of 300 fmols to 3,400 fmols.
  • the plurality of target polynucleotides is present at a concentration of 500 fmols to 3,400 fmols. In some embodiments, the plurality of target polynucleotides is present at a concentration of 750 fmols to 3,400 fmols. In some embodiments, the plurality of target polynucleotides is present at a concentration of 1,000 fmols to 3,400 fmols. In some embodiments, the plurality of target polynucleotides is present at a concentration of 1,000 fmols to 3,400 fmols. In some embodiments, the plurality of target polynucleotides is present at a concentration of 2,000 fmols to 3,400 fmols.
  • the plurality of target polynucleotides is present at a concentration of 50 fmols. In some embodiments, the plurality of target polynucleotides is present at a concentration of 3,400 fmols. In some embodiments, the plurality of target polynucleotides is present at a concentration of 500 fmols to 3,400 fmols.
  • the method of normalizing is performed for at least 2 samples (e.g., at least 3 samples, at least 4 samples, at least 5 samples, at least 6 samples, at least 7 samples, at least 8 samples, at least 9 samples, at least 10 samples, at least 25 samples, at least 50 samples, at least 75 samples, at least 100 samples, at least 150 samples, at least 200 samples, at least 300 samples, at least 384 samples, at least 400 samples, at least 500 samples, at least 600 samples, at least 700 samples, at least 800 samples, at least 900 samples, at least 1000 samples, 12372134.1 at least 1500 samples, at least 2000 samples, at least 3000 samples, or at least 5000 samples or more). In some embodiments, the method of normalizing is performed for 2-384 samples.
  • the method of normalizing is performed for 24-384 samples. In some embodiments, the method of normalizing is performed for 24-500 samples. In some embodiments, the method of normalizing is performed for 24-750 samples. In some embodiments, the method of normalizing is performed for 24-1000 samples. In some embodiments, the method of normalizing is performed for 24-2000 samples. In some embodiments, the method of normalizing is performed for 24-3000 samples. In some embodiments, a plurality of samples, each having an adapter sequence with a different index, are combined prior to next generation sequencing. In some embodiments, the samples comprise target polynucleotides at a pre- normalization concentration of within a 70-fold concentration of each other.
  • the samples comprise target polynucleotides at a pre-normalization concentration of within a 60-fold concentration of each other. In some embodiments, the samples comprise target polynucleotides at a pre-normalization concentration of within a 50-fold concentration of each other. In some embodiments, the samples comprise target polynucleotides at a pre- normalization concentration of within a 40-fold concentration of each other. In some embodiments, the samples comprise target polynucleotides at a pre-normalization concentration of within a 30-fold concentration of each other. In some embodiments, the samples comprise target polynucleotides at a pre-normalization concentration of within a 20-fold concentration of each other.
  • the samples comprise target polynucleotides at a pre- normalization concentration of within a 10-fold concentration of each other. In some embodiments, the samples comprise target polynucleotides at a pre-normalization concentration of within a 5-fold concentration of each other. In some embodiments, the samples comprise target polynucleotides at a pre-normalization concentration of within a 2-fold concentration of each other. In some embodiments, the samples comprise target polynucleotides at a pre- normalization concentration of within a 1.5-fold concentration of each other.
  • this disclosure provides a method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising: (i) obtaining the at least two samples, wherein the target polynucleotides of the at least two samples comprise: a first adapter sequence (e.g., an ILLUMINA, PACBIO, ELEMENT or ULTIMA adapter sequence (e.g., any one of SEQ ID 12372134.1 NOs: 1-4, 22, 27, 29, or 34)); (ii) contacting each of the at least two samples with primers that are complementary to a portion of the first adapter sequence thereby producing partially double stranded polynucleotides; (iii) binding the double stranded polynucleotides of each of the at least two samples with a predetermined amount of a ribonucleotide protein (RNP) complex, the RNP complex comprising: a dCas or a
  • RNP
  • the method comprises after (iii) and before (iv) combining the at least two samples and optionally removing unbound target polynucleotides from the combined samples.
  • this disclosure provides a method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising: (i) obtaining the at least two samples, wherein the target polynucleotides of the at least two samples comprise: a double stranded polynucleotide comprising an adapter sequence (e.g., an ILLUMINA, PACBIO, ELEMENT or ULTIMA adapter sequence (e.g., any one of SEQ ID NOs: 1-4, 22, 27, 29, or 34)); (ii) binding the double stranded polynucleotides of each of the at least two samples with a predetermined amount of a ribonucleotide protein (RNP) complex, the RNP complex comprising: a ribonucleot
  • the method comprises after (ii) and before (iii) combining the at least two samples and optionally removing unbound target polynucleotides from the combined samples.
  • this disclosure provides a method for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the method comprising: (i) obtaining the at least two samples, wherein the target polynucleotides of the at least two samples comprise: a first adapter sequence comprising: a proximal portion, and a distal portion comprising a sequence that binds to an adapter binding site of a next-generation sequencing platform during next-generation sequencing; and a second adapter sequence; (ii) contacting each of the at least two samples with primers that are complementary to the proximal portion of the first adapter sequence; (iii) extending the primers 12372134.1 in each of the at least two samples to produce reverse complements of the target polynucleotides
  • the method comprises after (iv) and before (v) combining the at least two samples and optionally removing unbound target polynucleotides from the combined samples. In some embodiments, removing unbound target polynucleotides from the combined samples comprising binding RNP complex to a solid phase (e.g., beads comprising a RNP binding tag) and washing the beads to remove unbound target polynucleotides.
  • a solid phase e.g., beads comprising a RNP binding tag
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples (e.g., two polynucleotide libraries) each comprising a target polynucleotide, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an ELEMENT, PACBIO, or ULTIMA adapter sequence (e.g., any one of SEQ ID NOs: 1-4, 22, 27, 29, or 34); (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a static region of the adapter sequence of the target polynucleotides of the sample, and (c) a predetermined concentration of a dCas or a dArgonaute; (iii) contacting the solution with a solid phase comprising a
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples (e.g., two polynucleotide libraries) each comprising a target polynucleotide, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an ELEMENT, PACBIO, or ULTIMA adapter sequence (e.g., any one of SEQ 12372134.1 ID NOs: 1-4, 22, 27, 29, or 34); (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a reverse complement of static region of the adapter sequence of the target polynucleotides of the sample, and (c) a predetermined concentration of a dCas or a dArgonaute; (iii) contacting the solution with
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples (e.g., two polynucleotide libraries) each comprising a target polynucleotide, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an ELEMENT, PACBIO, or ULTIMA adapter sequence (e.g., any one of SEQ ID NOs: 1-4, 22, 27, 29, or 34); (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the adapter sequence of the target polynucleotides of the sample, and (c) a predetermined concentration of a dCas or a dArgonaute; (iii) contacting the solution with a solid phase comprising a dCas
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples (e.g., two polynucleotide libraries) each comprising a target polynucleotide, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an ELEMENT OR PACBIO adapter sequence (e.g., any one of SEQ ID NOs: 1-4); (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a reverse complement of the adapter sequence of the target polynucleotides of the sample, and (c) a predetermined concentration of a dCas or a dArgonaute; (iii) contacting the solution with a solid phase comprising a dCas or a dArg
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index that is adjacent to a PAM; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a CRISPR- associated protein (Cas) guide RNA (gRNA) polynucleotide comprising a targeting region that is complementary to a reverse complement of a portion of the adapter sequence that is adjacent to the PAM of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas comprising an affinity tag, wherein the predetermined concentration of the dCas or the dArgonaute is cognate to the guide polynucle
  • the PAM is adjacent to the 5’ terminal of the index.
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index that is adjacent to a rPAM; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a CRISPR-associated protein (Cas) guide RNA (gRNA) polynucleotide comprising a targeting region that is complementary to a portion of the adapter sequence that is adjacent to the rPAM of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas comprising an affinity tag, wherein the predetermined concentration of the dCas or the dArg
  • the PAM is adjacent to the 3’ terminal of the index.
  • the target polynucleotide comprises a first adapter on its 3’ terminal end a second 12372134.1 adapter on its 5’terminal end.
  • the first adapter comprises a first index.
  • the second adapter comprises a second index.
  • the PAM is adjacent to the first index. In some embodiments, the PAM is adjacent to the second index.
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the index of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag, wherein the predetermined concentration of the dCas or the dArgonaute is cognate to the guide polynucleotide; (iii) contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable of binding to the affinity tag; (iv
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a reverse complement of the index of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag, wherein the predetermined concentration of the dCas or the dArgonaute is cognate to the guide polynucleotide; (iii) contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable of binding to the affinity
  • the target polynucleotide comprises a first adapter on its 3’ terminal end a second adapter on its 5’terminal end.
  • the first adapter comprises a first index.
  • the targeting region is complementary to the first index.
  • the targeting region is complementary to the second index.
  • the method of normalizing results in the at least two samples being more similar in polynucleotide concentration after the normalizing than before the normalizing. In some embodiments, the method of normalizing results in the at least two samples having a predetermined stoichiometric ratio of polynucleotides between the samples.
  • the predetermined ratio of polynucleotide may be 5:1, so after performing the method of normalizing, polynucleotides of a first sample may be about 5 times more abundant than polynucleotides of the second sample.
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the index of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag, wherein the predetermined concentration of the predetermined concentration of the
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the methods comprising, for each sample of the at least two samples: (i) obtaining the sample, wherein the target polynucleotides of the sample comprise an adapter sequence comprising an index; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a reverse complement of the index of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas or a dArgonaute 12372134.1 comprising an affinity tag, wherein the predetermined concentration of the dCas or the dArgonaute is cognate to the guide polynucleotide; (iii) contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable
  • the target polynucleotide comprises a first adapter on its 3’ terminal end a second adapter on its 5’ terminal end.
  • the first adapter comprises a first index.
  • the targeting region is complementary to the first index.
  • the targeting region is complementary to the second index.
  • the method of normalizing results in the at least two samples more similar in polynucleotide concentration after the normalizing than before the normalizing. In some embodiments, the method of normalizing results in the at least two samples having a predetermined stoichiometric ratio of polynucleotides between the samples.
  • the predetermined ratio of polynucleotide may be 5:1, so after performing the method of normalizing, polynucleotides of a first sample may be 5 times more abundant than polynucleotides of the second sample.
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, the methods comprising: (i) obtaining a mixture of the at least two samples, wherein the target polynucleotides of the at least two samples comprise an adapter sequence and an index, wherein different samples do not have the same index, and, for each sample, obtaining a guide polynucleotide comprising a targeting region that is complementary to the index, or a reverse complements of the index, of the sample; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of each a guide polynucleotide; and (c) a predetermined concentration of
  • a 12372134.1 “mixture of at least two samples” refers to the combination of polynucleotides of at least two samples.
  • the predetermined concentration of each a guide polynucleotide is different.
  • the different predetermined guide polynucleotide concentrations normalize the target polynucleotides of the different samples in a predetermined stoichiometric ratio. For example, in a method comprising obtaining a mixture of two samples (sample 1 and sample 2), the predetermined stoichiometric ratio of polynucleotides between sample 1 and sample 2 may be 1:2, 1:3, 1:4, 1:5, or 1:10.
  • the predetermined stoichiometric ratio is determined based on the desired sequencing depth of the polynucleotides of each sample. For example, a first sample may need more sequencing depth than a second sample because the first sample has a greater diversity of target polynucleotides.
  • normalizing comprises producing a double stranded PAM site on a single stranded target polynucleotide without requiring PCR amplification (e.g., FIGs. 2A- 2D).
  • PCR amplification is a popular strategy in the construction of NGS libraries, in particular because it allows for NGS sequencing on small amounts of sample. However, PCR amplification can be problematic for a number of NGS applications.
  • PCR amplification can introduce GC bias, which interferes with data analysis, such as the identification of novel single-nucleotide polymorphisms (SNPs).
  • SNPs single-nucleotide polymorphisms
  • a method of normalizing introduces a step of target polynucleotide denaturation and partial extension. This denaturation and extension is performed before a RNP binding complex is combined with the target nucleotides. This step begins with a sample having target polynucleotide comprising a 3’ and a 5’ adapter sequence (e.g., FIG. 2A). The reverse complement of the PAM site is encoded on the 5’ adapter.
  • the target polynucleotide e.g., FIG. 2B
  • the conditions include the presence of a DNA polymerase.
  • the elongated primer does not comprise a complete 3’ adapter (e.g., does not comprise a portion of the adapter that interacts with a sequencing device (e.g., a ILLUMINA flow cell or ULTIMA wafer binding sequence)), and therefore is not sequenced during next generation sequences. 12372134.1
  • sequencing results are not biased by amplified DNA (e.g., the elongated primer).
  • the partial primer can be complementary to a portion of the 5’ adapter sequence such that when the partial primer is extended it does not produce a reverse complement of the entire 5’ adapter resulting in an elongated polynucleotide that is not sequenced during next- generation sequencing because it has an incomplete 5’ adapter (e.g., the incomplete 5’ adapter does not comprise a portion of the adapter that interacts with a sequencing device (e.g., a ILLUMINA flow cell or ULTIMA wafer binding sequence)).
  • a sequencing device e.g., a ILLUMINA flow cell or ULTIMA wafer binding sequence
  • This partial primer method is suitable to use with numerous next-generation sequencing adapters including, but not limited to, ILLUMINA, ULTIMA, ELEMENT, and/or PACBIO sequencing adapters.
  • this disclosure provides a method of enriching a target polynucleotide in a sample, the method comprising: (i) obtaining the sample, wherein the target polynucleotide of the sample comprises an adapter sequence comprising an index; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the index; and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag, wherein the predetermined concentration of the dCas or the dArgonaute is cognate to the guide polynucleotide; (iii) contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable of binding to the affinity tag; (iv) separating the solution from the solid phase; and (v) extracting the target polynucleotides from the solid phase.
  • this disclosure provides a method of enriching a target polynucleotide in a sample, the method comprising: (i) obtaining the sample, wherein the target polynucleotide of the sample comprises an adapter sequence comprising an index; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a reverse complement of the index; and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag, wherein the predetermined concentration of the dCas or the dArgonaute is cognate to the guide polynucleotide; (iii) contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable of binding to the affinity tag; (iv) separating the solution from the solid phase; and (v) extracting the target polynucleotides from the solid phase
  • Enriching refers to a method of increasing the concentration of a target polynucleotide in a sample relative to the concentration of other polynucleotides in the sample.
  • 12372134.1 enriching a target polynucleotide may be performed by selectively extracting a target polynucleotide from the sample (e.g., using a Cas9 + guide RNA comprising a homology region that is complementary to the target polynucleotide) and then placing the target polynucleotide into a solution that is not the sample.
  • a method for normalizing and methods of enriching comprise obtaining one or more samples.
  • the sample may be obtained from any suitable source, including, but not limited to cells (e.g., eukaryotic or prokaryotic cells), tissues (e.g., brain, heart, lung, liver, kidney, adipose, skin, gall bladder, or breast), diseased tissues (e.g., tumor), bodily fluid (e.g., saliva, sweat, blood, urine, mucus, or cerebral spinal fluid), or from in vitro production.
  • cells e.g., eukaryotic or prokaryotic cells
  • tissues e.g., brain, heart, lung, liver, kidney, adipose, skin, gall bladder, or breast
  • diseased tissues e.g., tumor
  • bodily fluid e.g., saliva, sweat, blood, urine, mucus, or cerebral spinal fluid
  • obtaining a sample comprises obtaining polynucleotides of interest (e.g., extracting the polynucleotide of interest from a biological sample) and modifying the polynucleotides of interest to comprise adapters sequences (e.g., modifying the polynucleotides of interest to become target polynucleotides). Modifying the polynucleotides of interest to comprise adapter sequences may be performed using any suitable methods, including, but not limited to using a kit for attaching adapter sequences to polynucleotides described herein.
  • the sample comprises 1-100 nM of target polynucleotides.
  • the sample comprises 1-1000 nM of target polynucleotides.
  • producing a solution in the context of the method of normalization, refers to combining, the sample and the predetermined concentration of a guide polynucleotide and the predetermined concentration of a dCas or a dArgonaute in an aqueous solution (e.g., a buffer suitable for the method).
  • producing the solution comprises combining a pre-determined amount of dCas protein or dArgonaute protein and a predetermined amount of guide polynucleotide prior to combining with the sample.
  • producing a solution comprises combining a predetermined amount of a guide RNA polynucleotide that is RNA with a predetermined amount of dCas9 protein or dArgonaute protein. In some embodiments, producing a solution comprises combining a predetermined amount of a guide polynucleotide that is DNA with a predetermined amount of dArgonaute protein. In some embodiments, producing a solution comprises combining a predetermined amount of a guide RNA comprising a homology region of any one of SEQ ID NOs: 5-10 with a predetermined amount of dCas9 of SEQ ID NO: 13.
  • a “predetermined concentration” refers to a concentration (e.g., nanomolar) or amount (e.g., nanomoles) of a reagent (e.g., a guide polynucleotide, a dCas protein or a dArgonaute 12372134.1 protein) that has been selected for extracting a particular or consistent amount of target polynucleotides from a sample.
  • a reagent e.g., a guide polynucleotide, a dCas protein or a dArgonaute 12372134.1 protein
  • the same or similar amounts of a pre- determined concentration of a dCas protein or a dArgonaute protein is added to each sample that is to be normalized.
  • the same or similar amounts of a pre-determined concentration of a guide RNA polynucleotide is added to each sample that is to be normalized.
  • a predetermined concentration of a dCas protein or a dArgonaute protein is added to the sample, and a predetermined amount of guide RNA polynucleotide added to the sample is in excess of the predetermined amount of dCas protein or a dArgonaute protein added to the sample.
  • the predetermined amount of dCas protein or dArgonaute protein is 1-2000 femtomoles.
  • the predetermined amount of dCas protein or dArgonaute protein is 60-2000 femtomoles. In some embodiments, the predetermined amount of dCas protein or dArgonaute protein is 60-1000 femtomoles. In some embodiments, the predetermined amount of dCas protein or dArgonaute protein is 125-1000 femtomoles.
  • the predetermined amount of dCas protein or dArgonaute protein is at least 60 femtomoles (e.g., at least 60 femtomoles, at least 125 femtomoles, at least 250 femtomoles, at least 500 femtomoles, at least 750 femtomoles, or at least 1000 femtomoles). In some embodiments, the predetermined amount of dCas protein or dArgonaute protein is 50, 60, 125, 250, 750, 1000, 1500, or 2000 femtomoles.
  • the predetermined concentration of guide RNA polynucleotides is in excess to the predetermined concentration of dCas9 protein or dArgonaute protein.
  • ribonucleoprotein (RNP) complex e.g., the guide RNA, dCas protein or dArgonaute protein, and adapter sequence
  • RNP complex is present at in the sample at a concentration of 250 fmols to 14,000 fmols.
  • the RNP complex is present in the sample at a concentration of 300 fmols to 14,000 fmols.
  • the RNP complex is present in the sample at a concentration of 2,000 fmols to 14,000 fmols.
  • RNP complex is present in the sample at a concentration of 6,000 fmols to 14,000 fmols. In some embodiments, RNP complex is present in the sample at a concentration of 10,000 fmols to 14,000 fmols. In some embodiments, the RNP complex is present in the sample at a concentration of at least 250 fmols. In some embodiments, RNP complex is present in the sample at a concentration of at least 300 fmols. In some embodiments, the RNP complex is present in the sample at a concentration of at least 2,000 fmols. In some embodiments, RNP complex is present in the sample at a concentration of at least 6,000 fmols.
  • the RNP complex is present in the sample at a concentration of at least 10,000 fmols. In some embodiments, the RNP complex is present in the sample at a concentration of 250 fmols. In some embodiments, the RNP complex is present in the sample at a concentration of 300 fmols. In some embodiments, the RNP complex is present in the sample at a concentration of 2,000 fmols. In some embodiments, the RNP complex is present in the sample at a concentration of 6,000 fmols. In some embodiments, the RNP complex is present in the sample at a concentration of 10,000 fmols. In some embodiments, the RNP complex is present in the sample at a concentration of 14,000 fmols.
  • the method comprising producing a solution comprising a predetermined concentration of a dCas protein comprising an affinity tag or dArgonaute protein comprising an affinity tag as described herein.
  • the affinity tag binding molecule comprises a metal ion (e.g., Ni2+) and the affinity tag comprises a His Tag.
  • the affinity tag binding molecule comprises biotin and the affinity tag comprises avidin.
  • the affinity tag binding molecule comprises an anti-myc antibody and the affinity tag comprises a myc tag.
  • the affinity tag binding molecule and a corresponding affinity tag is selected from Table 1.
  • the dArgonaute or dCas of the method does not comprise an affinity tag.
  • the method comprises contacting the solution with a solid phase comprising a molecule that binds to the dCas or the dArgonaute (e.g., an antibody that binds to the dCas or dArgonaute).
  • the method comprises contacting the solution and a solid phase.
  • the solid phase comprises a molecule that binds to dArgonaute or dCas (e.g., an antibody or an affinity tag binding molecule).
  • the solid phase comprises a beads (e.g., a microparticle or a nanoparticle).
  • the bead is a metal bead, a polymer bead, a protein bead, or a lipid bead.
  • the bead e.g., metal bead
  • the bead comprises metal ions (e.g., Ni2+, Co2+, Cu2+, and/or Zn2+ ions) on the surface of the bead.
  • the metal bead is magnetic (e.g., paramagnetic, diamagnetic, or ferromagnetic).
  • the method comprises incubating the solid phase and the solution. In some embodiments, the incubation is at room temperature. In some embodiments, the incubation is at 30-40 ⁇ C.
  • the incubation is at 35-38 ⁇ C. In some 12372134.1 embodiments, the incubation is at or about 37 ⁇ C. In some embodiments, the incubation is at least 15 minutes (e.g., at least 30 minutes, at least 45 minutes, at least 60 minutes, or at least 2 hours). In some embodiments, the incubation is at least 60 minutes. In some embodiments, the incubation is 15 minutes to 2 hours. In some embodiments, the method further comprises separating the solution from the solid phase. “Separating” as used in the context of this method, refers to removing the solution from the solid phase or removing the solid phase from the solution.
  • separating is not a perfect separation, e.g., there may small amount of solution (e.g., less than about 1 microliter of solution) and solid phase that are still in contact with one another after separation.
  • the purpose of the separating step is to separate the unbound target polynucleotide of the solution from the target polynucleotides bound to the solid phase, which is part of normalizing concentration.
  • the method comprises washing the solid phase at least 1 (e.g., at least 2, at least 3, at least 4, or at least 5, or more times). In some embodiments, the method further comprises extracting the target polynucleotide from the solid phase.
  • the extracting comprising contacting the solid phase with a protease, which proteolyzes the Cas protein or Argonaute protein releasing the target polynucleotides.
  • the protease may be any suitable protease, including but not limited to trypsin, chymotrypsin, endoproteinase Lys-C, endoprotease AspN, endoprotease GluC, elastase, proteinase K, or papain. Corresponding protease reaction conditions and incubation times are known in the art.
  • the extracting comprising contacting the solid phase with a protein denaturing agent (e.g., a detergent, and organic solvent, or a chaotropic agent).
  • a protein denaturing agent e.g., a detergent, and organic solvent, or a chaotropic agent.
  • extracting comprising increasing the temperature of the solid phase (e.g., by warming a liquid in which the solid phase is located) to denature the Cas protein or Argonaute protein).
  • extracting comprises increasing or decreasing the pH of liquid in which the solid phase is located.
  • this disclosure provides methods for normalizing the concentration of target polynucleotides between at least two samples each comprising target polynucleotides, wherein the target polynucleotides that are not captured during normalization are digested.
  • the method comprises, for each sample of the at least two samples: (i) obtaining the sample (e.g., as described herein), wherein the target polynucleotides of the sample comprise an adapter sequence; (ii) producing a solution, the producing comprising combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the adapter sequence of the target polynucleotides of the sample, and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag; and (iii) contacting the solution with a nuclease (e.g., a catalytically active Cas or Argonaute protein 9) to normalize the concentration of target polynucleotides between the two or more samples (e.g., digest the target polynucleotides that are not bound to the solid phase).
  • a nuclease e.g.,
  • the method comprises contacting the solution with a nuclease.
  • the nuclease is an exonuclease.
  • the exonuclease is an exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, or exonuclease VIII.
  • the nuclease is a catalytically- active Cas RNP or Argonaute RNP.
  • the catalytically-active Cas RNP or Argonaute RNP comprises a guide polynucleotide that is complementary to an adapter sequence.
  • the Cas endonuclease is a Cas9 endonuclease.
  • the Cas endonuclease is a Cpf1, C2c1, C2c3, C2c2, CasX, or CasY endonuclease.
  • the catalytically-active is an Argonaute protein (e.g., an Argonaute endonuclease).
  • the Argonaute protein is a CbAgo endonuclease.
  • the Argonaute protein is a LrAgo, PfAgo, TtAgo, AaAgo, AfAgo, MjAgo, MpAgo, NgAgo, RsAgo, CpAgo, IbAgo, KmAgo, or SeAgo endonuclease.
  • the two more samples being normalized comprise target polynucleotides comprising a first adapter sequence and a second adapter sequence.
  • the guide polynucleotide targeting region is complementary to the first adapter sequence.
  • the method further comprises contacting the sample with a primer encoding a nucleic acid sequence that is complementary to a portion of the second adapter sequence and a polymerase (e.g., a DNA polymerase) in conditions sufficient for primer elongation.
  • a primer encoding a nucleic acid sequence that is complementary to a portion of the second adapter sequence and a polymerase (e.g., a DNA polymerase) in conditions sufficient for primer elongation.
  • this primer may be used to elongate a target polynucleotide such that first adapter sequence comprises a double stranded PAM for Cas (e.g., dCas) binding.
  • the primer is complementary to the proximal portion of an adapter sequence (e.g., the second adapter sequence).
  • elongating the primer to produce a double stranded 12372134.1 polynucleotide may not produce an elongated strand that is capable of being sequenced by next- generation sequence because the elongated strand does not comprise the distal portion of the second adapter sequence.
  • This may be advantageous because elongation (e.g., DNA amplification), can introduce artifacts (e.g., mutations) into polynucleotides that may bias or distort sequencing results.
  • the second adapter sequence is a 3’ adapter sequence. In some embodiments, the second adapter sequence is a 5’ adapter sequence.
  • Element Adapter 1 with the relevant Cas9 PAM site in bold 5’ GATCAGGTGAGGCTGCGACGACTTATGGCAATAGTTGACAAGCGGTAGCCTGCACA CCTTCCGACAT 3’ (SEQ ID NO: 1) Element guide RNA 1A (CGG PAM site) 5’ UUAUGGCAAUAGUUGACAAG 3’ (SEQ ID NO: 5) Element Adapter 2 There are 2 relevant PAM sites AGG, GGG, 5’ CATGTAATGCACGTACTTTCAGGGTATATTGGTGCGTGCTGGATTGGCTCACCAGAC ACCTTCCGACA 3’ (SEQ ID NO: 2) Element guide RNA 2A 5’ CAUGUAAUGCACGUACUU
  • index begins with a PAM site (bold).
  • the guide RNA homology region for the above sequence could be: 5’ ACGUCUGAACUCCAGUCACA 3’ (SEQ ID NO: 28)
  • PAMs adjacent to index sequences allows normalizing libraries which do not normally have a PAM site built in. By making the PAM site part of the index sequence the sequencing performance will not be affected but normalization will be possible for non-PAM-site containing adapter types. In general, the idea is to introduce a constant PAM site into the index sequence of an adapter.
  • dCas9 RNP solutions was formed by combining 100 picomoles of dCas9 with 150 picomoles of sgRNA (either UG_sgRNA_1, UG_sgRNA_2 or UG_sgRNA_3) in the presence of 50 mM HEPES pH 6.5, 100 mM NaCl, 5 mM MgCl2 and 0.02% Tween-20 and incubating for 20 minutes at 25°C in a volume of 100 ul.
  • the resulting 1 picomole/ ul (1000 nM) RNP solutions were diluted to 25 nM with 50 mM HEPES pH 6.5, 100 mM NaCl, 5 mM MgCl2 and 0.02% Tween-20.
  • ULTIMA Genomics library containing the “A” adapter sequences was prepared from human genomic DNA (genotype NA12878), using the Watchmaker Genomics Library Preparation kit with Fragmentation, followed by PCR amplification.
  • the library was made up to a molar concentration of 50 nM (50 femtomoles/ ul) with water.
  • Library capture using limiting amounts of RNP combined with excess library 500 femtomoles of the library (10 ul) were combined with 250 fmoles of each RNP and incubated at 37C for 15 minutes to bind RNP to library molecules.
  • Dynabeads His-Tag Isolation and Pulldown beads (Thermo Fisher Scientific) were added to each binding reaction, followed by incubation at room temperature for 5 minutes, to bind the library:RNP complexes to the beads.
  • Beads were washed with 100 ul of 50 mM HEPES pH 6.5, 100 mM NaCl, 5 mM MgCl2 and 0.04% Tween-20. Libraries were eluted by resuspending the beads in 10 mM Tris-Cl pH 8.0, 0.1% SDS and incubated at 70C for 5 minutes.

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Abstract

La présente invention concerne des procédés et des compositions pour normaliser la concentration de polynucléotides et enrichir des polynucléotides à partir d'un échantillon.
PCT/US2025/026068 2024-04-24 2025-04-23 Procédés de normalisation de bibliothèque de séquençage Pending WO2025226876A1 (fr)

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