Methods and compositions for sequencing library normalization
Disclosed herein are methods and compositions for normalizing polynucleotide concentration. Normalizing may be accomplished using polynucleotide binding proteins (e.g., catalytically inactive CRISPR protein or catalytically inactive Argonaute proteins). The polynucleotide binding proteins may bind to the adapter sequences of a target polynucleotide. Thus, adding the same amount of a polynucleotide binding proteins to different samples and then extracting the polynucleotide protein is shown herein to extract similar amounts of target polynucleotides from the different samples.
1. 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:
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 the target polynucleotides with primers that are complementary to the proximal portion of the first adapter sequence;
(iii) extending the primers to produce reverse complements of the target polynucleotides that do not comprise a reverse complement of the distal portion of the first adapter sequence, thereby producing partially double stranded polynucleotides comprising a double stranded second adapter sequence;
(iv) producing a solution, the producing comprising combining: (a) the partially double stranded polynucleotides, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a strand of the double stranded second adapter sequence; and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag, wherein the dCas or the dArgonaute is cognate to the guide polynucleotide;
(v) contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable of binding to the affinity tag;
(vi) separating the solution from the solid phase; and
(vii) extracting the target polynucleotides from the solid phase to normalize the concentration of target polynucleotides between the two or more samples.
2. The method of claim 1 , wherein the targeting region is complementary to a static region of the proximal portion.
3. The method of claim 1 , wherein the guide polynucleotide comprises a CRISPR-associated protein (Cas) guide RNA (gRNA) polynucleotide or an Argonaute guide polynucleotide.
4. The method of claim 3 , wherein the Cas gRNA polynucleotide targeting region is a homology region.
5. The method of claim 4 , wherein the Cas gRNA polynucleotide comprises a homology region of any one of SEQ ID NOs: 1-6, 9-19 and 21-26.
6. The method of claim 5 , wherein the Cas gRNA polynucleotide comprises a homology region of SEQ ID NO: 1.
7. The method of claim 1 , wherein the guide polynucleotide comprises a modified nucleic acid.
8. The method of claim 1 , wherein the dCas is a catalytically-inactive Cpf1, C2c1, C2c3, C2c2, CasX, Cas9, or CasY protein.
9. The method of claim 8 , wherein the dCas protein comprises an amino acid sequence of SEQ ID NO: 37.
10. The method of claim 1 , wherein the affinity tag binding molecule comprises Ni2+and the affinity tag comprises a His Tag.
11. The method of claim 1 , wherein extracting the target polynucleotides from the solid phase comprises combining the solid phase and a protease.
12. The method of claim 1 , wherein normalizing comprises making the concentration of the of target polynucleotides between the at least two samples within 15% of one another after normalization.
13. The method of claim 1 , wherein the method does not comprise amplifying the target polynucleotides of the at least two samples using polymerase chain reaction.
14. The method of claim 1 , wherein the sequence that binds to the adapter binding site is a flow cell binding sequence.
15. 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:
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 the target polynucleotides with primers that are complementary to the proximal portion of the first adapter sequence;
(iii) extending the primers to produce reverse complements of the target polynucleotides that do not comprise a reverse complement of the distal portion of the first adapter sequence, thereby producing partially double stranded polynucleotides comprising a double stranded second adapter sequence;
(iv) producing a solution, the producing comprising combining: (a) the partially double stranded polynucleotides, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to a strand of the double stranded second adapter sequence; and (c) a predetermined concentration of a dCas or a dArgonaute comprising an affinity tag; and
(v) contacting the solution with a pre-determined amount of a catalytically active Cas or Argonaute protein to normalize the concentration of target polynucleotides between the two or more samples.
16. 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 in each of the at least two samples to produce reverse complements of the target polynucleotides that do not comprise a reverse complement of the distal portion of the first adapter sequence, thereby producing partially double stranded polynucleotides comprising a double stranded second adapter sequence;
(iv) 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 dArgonaute, and a guide polynucleotide that comprises a targeting region which is complementary to a strand of the double stranded second adapter sequence;
and (v) extracting the target polynucleotides from the RNP complex to normalize the concentration of target polynucleotides between the at least two samples.
17. The method of claim 16 , wherein the RNP complex comprises a dCas protein and a Cas guide RNA.
18. The method of claim 17 , wherein the Cas guide RNA comprises a homology region of any one of SEQ ID NOs: 1-26, and the dCas protein is dCas9.
19. The method of claim 18 , wherein the Cas gRNA polynucleotide comprises a homology region of SEQ ID NO: 1.
20. The method of claim 16 , wherein extracting the target polynucleotides from the RNP complex comprising contacting the RNP with a solid phase comprising an RNP complex binding molecule.
21. The method of claim 20 , wherein extracting the target polynucleotides from the RNP complex comprises washing the solid phase.
22. The method of claim 16 , wherein the method does not comprise amplifying the target polynucleotides of the at least two samples using polymerase chain reaction.
23. The method of claim 16 , wherein obtaining the at least two samples comprises:
(i) obtaining at least fifty samples, wherein the target polynucleotides of the at least fifty 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 fifty samples with primers that are complementary to the proximal portion of the first adapter sequence;
iii) extending the primers in each of the at least fifty samples to produce reverse complements of the target polynucleotides that do not comprise a reverse complement of the distal portion of the first adapter sequence, thereby producing partially double stranded polynucleotides comprising a double stranded second adapter sequence;
(iv) binding the double stranded polynucleotides of each of the at least fifty samples with a predetermined amount of a ribonucleotide protein (RNP) complex, the RNP complex comprising: a dCas9, a guide RNA that comprises a homology which is complementary to a strand of the double stranded second adapter sequence, and an affinity tag; and
(v) extracting the target polynucleotides from the RNP complex to normalize the concentration of target polynucleotides between the at least fifty samples, the extracting comprising contacting the RNP with a solid phase comprising an affinity tag binding molecule and washing the solid phase.
24. The method of claim 16 , wherein the sequence that binds to the adapter binding site is a flow cell binding sequence.
25. The method of 20 , wherein the RNP comprises an affinity tag and the RNP complex binding molecule comprises an affinity tag binding molecule.