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 adaptor, a barcode, a capture domain, a sequence that is complementary to all or a portion of the nucleic acid analyte sequence, and a second adaptor, 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;
(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; and
(d) amplifying the double-stranded circularized nucleic acid using a first primer and a second primer to generate a double-stranded member of the nucleic acid library lacking all, or a portion of, the nucleic acid analyte sequence, wherein:
the first primer comprises: (i) a sequence substantially complementary to a 3′ region of the nucleic acid analyte sequence, and (ii) a first functional domain comprising a sequence for attachment to a flow cell; and
the second primer comprises: (i) a sequence substantially complementary to a 5′ region of the nucleic acid analyte sequence, and (ii) a second functional domain comprising a primer sequence to amplify the 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 adaptor, the barcode, the capture domain, the sequence complementary to all or a portion of the nucleic acid analyte sequence, and the second adaptor, 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 sequence comprising the first functional domain, in 3′ to 5′ direction; and wherein the second primer comprises (i) the sequence substantially complementary to the 5′ region of the nucleic acid analyte sequence, and (ii) the sequence comprising the second functional domain, in a 3′ to 5′ direction.
5. The method of claim 1 , wherein the barcode is a spatial barcode or a cell barcode.
6. The method of claim 1 , wherein ligating in step (c) is performed using a DNA ligase or using template mediated ligation.
7. The method of claim 1 , wherein the nucleic acid library is a DNA library or a cDNA library.
8. The method of claim 1 , wherein the nucleic acid analyte sequence encodes an immune cell receptor.
9. The method of claim 8 , wherein the nucleic acid analyte sequence encodes a constant region of the immune cell receptor.
10. The method of claim 9 , wherein the nucleic acid analyte sequence further encodes a variable region of the immune cell receptor.
11. The method of claim 10 , 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.
12. The method of claim 8 , wherein the immune cell receptor is a B cell receptor.
13. The method of claim 12 , 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 further comprises one or 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.
14. The method of claim 12 , 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.
15. The method of claim 12 , wherein the B cell receptor comprises an immunoglobulin heavy chain.
16. The method of claim 15 , wherein the immunoglobulin heavy chain comprises a variable region comprising a CDR3 of the immunoglobulin heavy chain, and 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.
17. The method of claim 8 , wherein the immune cell receptor is a T cell receptor.
18. The method of claim 17 , 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.
19. The method of claim 17 , 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.
20. The method of claim 1 , wherein the method further comprises amplifying the double-stranded member of the nucleic acid library lacking all, or a portion of, the nucleic acid analyte sequence using a third primer and a fourth primer, wherein:
the third primer is substantially complementary to the first functional domain, and
the fourth primer is substantially complementary to the second functional domain.
21. The method of claim 10 , wherein the method further comprises:
determining (i) all or a portion of the sequence encoding the variable region or a complement thereof, and (ii) the sequence of the barcode or a complement thereof.
22. The method of claim 21 , wherein the determining step comprises sequencing (i) all or a portion of the sequence encoding the variable region or a complement thereof, and (ii) the sequence of the barcode or a complement thereof.
23. The method of claim 22 , wherein the sequencing is performed by sequence-by-synthesis, sequence-by-ligation, or sequence-by-hybridization.
24. The method of claim 21 , wherein the nucleic acid analyte sequence was released from a biological sample, and the method further comprises:
determining the location of the nucleic acid analyte sequence 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 generating the double-stranded member of the nucleic acid library comprises:
contacting the nucleic acid analyte sequence with a capture probe comprising the first adaptor, the barcode, and the capture domain, wherein the capture domain binds specifically to a sequence present in the nucleic acid analyte sequence;
extending an end of the capture probe using the nucleic acid analyte sequence specifically bound to the capture domain as a template, thereby generating an extended capture probe; and
adding the second adaptor 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 a 3′ end of the capture probe is extended.
28. The method of claim 26 , 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 formalin-fixed paraffin-embedded tissue section or a fresh, frozen tissue section.
30. The method of claim 1 , wherein the nucleic acid analyte sequence is RNA, mRNA, DNA, or genomic DNA.