Methods of single-cell barcoding and sequencing
Provided herein are methods for immune repertoire sequencing and single cell barcoding. In some aspects, such methods may comprise producing a plurality of cDNAs from a plurality of polynucleotides, wherein at least one polynucleotide encodes a heavy and/or light chain, from a plurality of immune cells from a biological sample. Producing a plurality of cDNAs may comprise reverse transcription and template switching, such as to form a plurality of uniquely barcoded cDNAs. Methods described herein further comprise amplifying the plurality of uniquely barcoded cDNAs, thereby forming a library, and sequencing one or more of the sequences of the library.
1. A method comprising:
producing a plurality of cDNAs from a plurality of polynucleotides, wherein at least one polynucleotide encodes a heavy and/or light chain, from a plurality of immune cells from a biological sample, the producing comprising:
(i) reverse transcribing a sequence complementary to a sequence downstream of a variable region of the at least one polynucleotide encoding a heavy and/or light chain, using:
(A) a first plurality of primers, and
(B) a reverse transcriptase comprising a non-template terminal transferase activity, wherein 3 or more identical non-template nucleotides are added to a 3′ end of the cDNAs, and
(ii) template switching, using a plurality of template switch polynucleotides, each comprising:
(A) a unique barcode,
(B) a first primer binding site 5′ to the unique barcode, and
(C) a 3′ end region complementary to the 3 or more identical non-template nucleotides,
thereby forming a plurality of uniquely barcoded cDNAs; wherein the reverse transcribing and the template switching are performed in an emulsion;
wherein the method optionally further comprises:
(a) amplifying the plurality of uniquely barcoded cDNAs, thereby forming a library of uniquely barcoded sequences comprising a variable region of the heavy (V H ) or light (V L ) chain polynucleotides;
and wherein the method further optionally comprises:
(b) sequencing one or more of the sequences of the library.
2. The method of claim 1 , further comprising amplifying the plurality of uniquely barcoded cDNAs with:
(i) a first primer comprising
(A) a region that binds to a complement of the same region sequence of the cDNAs, and
(B) a 5′ second primer binding site; and
(ii) a second primer comprising a region that binds to the complement of the first primer binding site.
3. The method of claim 2 , wherein the plurality of uniquely barcoded cDNAs are further amplified with:
(i) a third primer comprising a 5′ first universal primer binding site; and
(ii) a fourth primer comprising a 5′ second universal primer binding site.
4. The method of claim 3 , further comprising sequencing one or more of the sequences of the library with a primer set comprising a first primer complementary to the first universal primer binding site and a second primer complementary to the second universal primer binding site.
5. The method of claim 3 , wherein the third primer comprises a sample barcode sequence 3′ to the first universal primer binding site or the fourth primer comprises a sample barcode sequence 3′ to the second universal primer binding site.
6. The method of claim 4 , wherein the sequencing comprises high-throughput sequencing.
7. The method of claim 2 , wherein the library comprises a plurality of V H or V L sequences representing an immune state of the biological sample.
8. The method of claim 7 , further comprising sequencing one or more of the sequences of the library and determining a variance of the V H or V L sequences from those of a germ line sequence.
9. The method of claim 7 , further comprising sequencing one or more of the sequences of the library and determining:
(i) a total number of unique heavy chain polynucleotides,
(ii) a total number of unique light chain polynucleotides,
(iii) a frequency of a heavy chain polynucleotide, or
(iv) a frequency of a light chain polynucleotide.
10. The method of claim 9 , further comprising selecting a therapeutic antibody based on
(i) a total number of unique heavy or light chain polynucleotides,
(ii) a frequency of a heavy or light chain polynucleotide, or
(iii) a variance of a V H or V L sequence from that of a germ line sequence.
11. The method of claim 4 , further comprising producing a consensus sequence from sequences in the library comprising a same unique barcode.
12. The method of claim 4 , wherein sequencing errors are minimized, eliminated, or less than 0.01%.
13. The method of claim 2 , wherein amplification bias is minimized, eliminated, or less than 0.01%.
14. The method of claim 4 , further comprising comparing the sequences of the library from the biological sample to sequences of a library from a biological sample taken from a same subject at a different time point.
15. The method of claim 1 , wherein the biological sample is from a subject with a condition or disease.
16. The method of claim 1 , wherein the sequence downstream of the variable region of the polynucleotides encoding a heavy and/or light chain comprises a polyA sequence.
17. The method of claim 1 , wherein a region of the first plurality of primers that is complementary to sequence downstream of the variable region of the polynucleotides encoding a heavy and/or light chain comprises a poly-T sequence.
18. The method of claim 1 , wherein the sequence downstream of the variable region of the polynucleotides encoding a heavy and/or light chain comprises a heavy or light chain constant region sequence.
19. The method of claim 4 , wherein the biological sample is from a subject with a condition or disease and wherein the method further comprises comparing the sequences of the library from the biological sample to sequences of a library from a biological sample taken from a subject without the condition or disease.