Nucleic acid library methods

1 . A method for removing all or a portion of an immune cell receptor 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 spatial barcode, a capture domain, a sequence that is complementary to all or a portion of the immune cell receptor 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 immune cell receptor sequence, wherein:

the first primer comprises: (i) a sequence substantially complementary to a 3′ region of the immune cell receptor 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 immune cell receptor 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 immune cell receptor 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 spatial barcode, a unique molecular identifier, the capture domain, the sequence complementary to all or a portion of the immune cell receptor sequence, and the second adaptor, in a 5′ to 3′ direction.

3 . The method of claim 1 , wherein the first primer comprises: (i) the sequence substantially complementary to the 3′ region of the immune cell receptor 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 immune cell receptor sequence, and (ii) the sequence comprising the second functional domain, in a 3′ to 5′ direction.

4 . The method of claim 1 , wherein ligating in step (c) is performed using a DNA ligase or using template mediated ligation.

5 . The method of claim 1 , wherein the nucleic acid library is a DNA library or a cDNA library.

6 . The method of claim 1 , wherein the immune cell receptor sequence encodes a constant region of the immune cell receptor and/or the immune cell receptor sequence encodes a variable region of the immune cell receptor.

7 . The method of claim 6 , wherein the sequence that is complementary to all or a portion of the immune cell receptor 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 immune cell receptor sequence encoding the variable region of the immune cell receptor.

8 . The method of claim 1 , wherein the immune cell receptor is a B cell receptor, 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.

9 . The method of claim 8 , 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.

10 . The method of claim 8 , wherein the B cell receptor comprises an immunoglobulin heavy chain, 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.

11 . The method of claim 1 , wherein the immune cell receptor is a T cell receptor 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.

12 . The method of claim 11 , 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.

13 . 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 immune cell receptor 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.

14 . The method of claim 6 , 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 spatial barcode or a complement thereof.

15 . The method of claim 14 , 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 spatial barcode or a complement thereof and wherein the sequencing is performed by sequence-by-synthesis, sequence-by-ligation, or sequence-by-hybridization.

16 . The method of claim 14 , wherein the immune cell receptor sequence was released from a biological sample, and the method further comprises:

determining the location of the immune cell receptor sequence in the biological sample using the determined sequences of (i) and (ii).

17 . The method of claim 1 , further comprising generating the double-stranded member of the nucleic acid library, wherein generating the double-stranded member of the nucleic acid library comprises:

contacting the immune cell receptor sequence with a capture probe comprising the first adaptor, the spatial barcode, and the capture domain, wherein the capture domain binds specifically to a sequence present in the immune cell receptor sequence;

extending an end of the capture probe using the immune cell receptor 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.

18 . The method of claim 17 , wherein a 3′ end of the capture probe is extended and the second adapter is added to a 5′ end of the extended capture probe.

19 . The method of claim 16 , 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.

20 . The method of claim 1 , wherein the immune cell receptor sequence is RNA, mRNA, DNA, or genomic DNA.