High-throughput immune sequencing
Methods and compositions for determining and/or monitoring the immune state of an individual are provided.
1. A method comprising:
(a) linking a polynucleotide sequence encoding a heavy chain variable region (V H ) and a polynucleotide sequencing encoding a light changing variable region (V L ) from a single cell to form paired sequences;
(b) sequencing a polynucleotide amplified from the paired sequences, thereby forming paired sequence reads;
(c) selecting a therapeutic antibody candidate comprising a V H and a V L encoded by at least one sequence pair linked in (a); and
(d) screening the therapeutic antibody candidate for a property, wherein the property is selected from the group consisting of functional specificity, affinity and neutralization ability;
wherein (a) is performed for a plurality of polynucleotide sequences encoding heavy chain variable regions (V H ) and a plurality of polynucleotide sequences encoding light chain variable regions (V L ) each from a single cell to form a plurality of paired sequences, each representing a sequence pair from an individual cell, and wherein selecting is based on a frequency of each of two or more paired V H and V L sequences from among the plurality of paired sequences and a variance from a reference library for at least one of the two or more paired V H and V L sequences.
2. The method of claim 1 , wherein the linking is a physical linking.
3. The method of claim 1 , further comprising cloning the therapeutic antibody directly into surface-display technology.
4. The method of claim 1 , wherein the therapeutic antibody is a neutralizing antibody.
5. The method of claim 1 , wherein the therapeutic antibody is a rapid response antibody.
6. The method of claim 1 , further comprising determining an isotype of the therapeutic antibody.
7. The method of claim 1 , wherein the polynucleotide amplified from the paired sequences is amplified by PCR with non-specific primers, degenerate primers, or specific primers.
8. The method of claim 1 , wherein the polynucleotide amplified from the paired sequences is amplified by PCR with specific primer sets that hybridize to the heavy and light chains of: B-cells, T-cells, or B-cells and T-cells.
9. The method of claim 1 , wherein the polynucleotide amplified from the paired sequences is amplified by PCR with two sets of primers after (a), wherein the first set of primers hybridizes to V regions and the second set of primers hybridizes to C regions.
10. The method of claim 1 , further comprising generating a database of paired V H and V L sequences.
11. The method of claim 10 , wherein selecting comprises aligning sequence data from the database against known or expected V, D and J segments in: a personal database, a NCBI database, or an IMGT database.
12. The method of claim 1 , wherein the cell is a B-cell.
13. The method of claim 1 , wherein the therapeutic antibody is fully human.
14. The method of claim 1 , wherein the polynucleotide amplified from the paired sequences is DNA.
15. The method of claim 1 , wherein the polynucleotide amplified from the paired sequences comprises sequence adaptors compatible with next-generation high-throughput sequencing.
16. The method of claim 1 , wherein the sequencing is next-generation high-throughput sequencing.
17. The method of claim 1 , wherein the paired sequences encoding a V H and a V L are bar-coded.
18. The method of claim 1 , wherein in (c), a coefficient of variation of about 0.5 occurs at clone frequencies of <10 −4 -10 −3 .
19. The method of claim 1 , wherein the variance is a variance of an alignment of a paired sequence to a sequence of the reference library.
20. The method of claim 1 , wherein in (c), a coefficient of variation approaches 1 at a sampling level of 10 5 .
21. The method of claim 1 , further comprising clustering the paired sequences into unique clones.
22. The method of claim 4 , further comprising identifying an antigen that interacts with the therapeutic antibody as a biomarker for a disease.
23. The method of claim 1 , wherein each paired sequence of the plurality of paired sequences represents a sequence pair from an individual cell of a plurality of individually isolated single cells.
24. The method of claim 23 , wherein the single cell is isolated from a single subject.
25. The method of claim 1 , further comprising isolating and purifying the therapeutic antibody candidate.