Nucleic acid library methods
Provided herein are methods, compositions, and kits for removing a portion of a sequence in a member of a nucleic acid library.
1. A method for removing all or a portion of a nucleic acid analyte sequence from a double-stranded member of a nucleic acid library, wherein a single strand of the double-stranded member of the nucleic acid library comprises: a first adapter, a barcode, a capture domain, a sequence that is complementary to all or a portion of the nucleic acid analyte sequence, and a second adapter, wherein the method comprises:
(a) ligating to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence;
(b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end of the double-stranded member of the nucleic acid library;
(c) ligating the ends of the double-stranded member of the nucleic acid library of step (b) to generate a first double-stranded circularized nucleic acid;
(d) amplifying the first double-stranded circularized nucleic acid using a first primer and a second primer to generate a first double-stranded nucleic acid product comprising a second restriction endonuclease recognition sequence at each end of the first double-stranded nucleic acid product, wherein:
the first primer comprises: (i) a sequence substantially complementary to a 3′ region of the nucleic acid analyte sequence and (ii) the second restriction endonuclease recognition sequence; and
the second primer comprises: (i) a sequence substantially complementary to a 5′ region of the nucleic acid analyte sequence, and (ii) the second restriction endonuclease recognition sequence;
(e) contacting the first double-stranded nucleic acid product with a second restriction endonuclease that cleaves the second restriction endonuclease recognition sequence at each end of the first double-stranded nucleic acid product;
(f) ligating the ends of the first double-stranded nucleic acid product of step (e) to generate a second double-stranded circularized nucleic acid; and
(g) amplifying the second double-stranded circularized nucleic acid using a third primer comprising a sequence that is substantially complementary to the first adapter and a fourth primer comprising a sequence that is substantially complementary to the second adapter, to generate a double-stranded member of the nucleic acid library lacking all, or a portion of, the nucleic acid analyte sequence.
2. The method of claim 1 , wherein the single-strand of the double-stranded member of the nucleic acid library comprises the first adapter, the barcode, the capture domain, the sequence that is complementary to all or a portion of the nucleic acid analyte sequence, and the second adapter, in a 5′ to 3′ direction.
3. The method of claim 2 , wherein the single-strand of the double-stranded member of the nucleic acid library further comprises a unique molecular identifier (UMI) disposed between the barcode and the capture domain.
4. The method of claim 1 , wherein the first primer comprises (i) the sequence substantially complementary to the 3′ region of the nucleic acid analyte sequence and (ii) the second restriction endonuclease recognition sequence, in a 3′ to 5′ direction.
5. The method of claim 1 , wherein the second primer comprises (i) the sequence substantially complementary to the 5′ region of the nucleic acid analyte sequence and (ii) the second restriction endonuclease recognition sequence, in a 3′ to 5′ direction.
6. The method of claim 1 , wherein the barcode is a cell barcode or a spatial barcode.
7. The method of claim 1 , wherein the ligating in step (c) and/or step (f) is performed using a ligase or using template mediated ligation.
8. The method of claim 7 , wherein the ligase is a DNA ligase, optionally wherein the DNA ligase is T4 ligase.
9. The method of claim 1 , wherein the nucleic acid library is a DNA library or a cDNA library.
10. The method of claim 1 , wherein the nucleic acid analyte sequence encodes an immune cell receptor.
11. The method of claim 10 , wherein the nucleic acid analyte sequence encodes a constant region of the immune cell receptor.
12. The method of claim 11 , wherein the nucleic acid analyte sequence further encodes a variable region of the immune cell receptor.
13. The method of claim 12 , wherein the sequence that is complementary to all or a portion of the nucleic acid analyte sequence encoding the constant region of the immune cell receptor is positioned 5′ relative to the sequence that is complementary to all or a portion of the nucleic acid analyte sequence encoding the variable region of the immune cell receptor.
14. The method of claim 12 , wherein the sequence that is complementary to all or a portion of the nucleic acid analyte sequence encoding the constant region of the immune cell receptor is positioned 3′ relative to the sequence that is complementary to all or a portion of the nucleic acid analyte sequence encoding the variable region of the immune cell receptor.
15. The method of claim 12 , wherein the immune cell receptor is a B cell receptor.
16. The method of claim 15 , wherein the B cell receptor comprises an immunoglobulin kappa light chain and wherein the variable region comprises a CDR3 of the immunoglobulin kappa light chain, or
wherein the variable region comprises or one both of CDR1 and CDR2 of the immunoglobulin kappa light chain, or
wherein the variable region further comprises a full-length variable domain of the immunoglobulin kappa light chain.
17. The method of claim 15 , wherein the B cell receptor comprises an immunoglobulin lambda light chain, and wherein the variable region comprises a CDR3 of the immunoglobulin lambda light chain, or
wherein the variable region further comprises one or both of CDR1 and CDR2 of the immunoglobulin lambda light chain, or
wherein the variable region further comprises a full-length variable domain of the immunoglobulin lambda light chain.
18. The method of claim 15 , wherein the B cell receptor comprises an immunoglobulin heavy chain, and wherein the variable region comprises a CDR3 of the immunoglobulin heavy chain, or wherein the variable region further comprises one or both of CDR1 and CDR2 of the immunoglobulin heavy chain, or wherein the variable region further comprises a full-length variable domain of the immunoglobulin heavy chain.
19. The method of claim 12 , wherein the immune cell receptor is a T cell receptor.
20. The method of claim 19 , wherein the T cell receptor comprises a T cell receptor alpha chain and wherein the variable region comprises a CDR3 of the T cell receptor alpha chain, or
wherein the variable region further comprises one or both of CDR1 and CDR2 of the T cell receptor alpha chain, or
wherein the variable region further comprises a full-length variable domain of the T cell receptor alpha chain.
21. The method of claim 19 , wherein the T cell receptor comprises a T cell receptor beta chain and wherein the variable region comprises a CDR3 of the T cell receptor beta chain, or
wherein the variable region further comprises one or both of CDR1 and CDR2 of the T cell receptor beta chain, or
wherein the variable region further comprises a full-length variable domain of the T cell receptor beta chain.
22. The method of claim 12 , wherein the method further comprises:
(h) determining (i) all or a portion of the sequence encoding the variable region of the immune cell receptor or a complement thereof, and (ii) the sequence of the barcode or a complement thereof.
23. The method of claim 22 , wherein the determining in step (h) comprises sequencing (i) all or a portion of the sequence encoding the variable region of the immune cell receptor or a complement thereof, and (ii) the sequence of the barcode or a complement thereof.
24. The method of claim 22 , wherein the nucleic acid analyte was released from a biological sample, and the method further comprises:
determining a location of the nucleic acid analyte in the biological sample using the determined sequences of (i) and (ii).
25. The method of claim 1 , further comprising generating the double-stranded member of the nucleic acid library.
26. The method of claim 25 , wherein the step of generating the double-stranded member of the nucleic acid library comprises:
contacting the nucleic acid analyte with a capture probe comprising the first adapter, the barcode, and the capture domain, wherein the capture domain binds specifically to a sequence present in the nucleic acid analyte;
extending an end of the capture probe using the nucleic acid analyte specifically bound to the capture domain as a template, thereby generating an extended capture probe; and
adding the second adapter to an end of the extended capture probe, thereby generating the double-stranded member of the nucleic acid library.
27. The method of claim 26 , wherein the capture probe comprises the first adapter, the barcode, and the capture domain in a 5′ to 3′ direction.
28. The method of claim 27 , wherein a 3′ end of the capture probe is extended, and optionally, wherein the second adapter is added to a 5′ end of the extended capture probe.
29. The method of claim 24 , wherein the biological sample is a tissue sample or a tissue section, and optionally, wherein the tissue section is a formalin-fixed paraffin-embedded tissue section or the tissue section is a fresh, frozen tissue section.
30. The method of claim 1 , wherein the nucleic acid analyte sequence is RNA, mRNA, DNA, or genomic DNA.