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1. WO2017177308 - HYBRID-CAPTURE SEQUENCING FOR DETERMINING IMMUNE CELL CLONALITY

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[ EN ]

CLAIMS:

1. A method of capturing a population of T-Cell receptor and/or immunoglobulin sequences with variable regions within a patient sample, said method comprising: extracting/preparing DNA fragments from the patient sample;

ligating a nucleic acid adapter to the DNA fragments, the nucleic acid adapter suitable for recognition by a pre-selected nucleic acid probe;

capturing DNA fragments existing in the patient sample using a collection of nucleic acid hybrid capture probes, wherein each capture probe is designed to hybridize to a known V gene segment and/or a J gene segment within the T cell receptor and/or immunoglobulin genomic loci.

2. The method of claim 1 , further comprising sequencing the captured DNA fragments, wherein the sequencing can be used to determine clonotypes within the patient sample.

3. The method of claim 1 , wherein said sequencing is optimized for short read sequencing.

4. The method of claim 2, further comprising amplifying the population of sequences using nucleic acid amplification probes/oligonucleotides that recognize the adapter prior to said sequencing.

5. The method of any one of claims 1-4, further comprising fragmenting DNA extracted from the patient sample to generate the DNA fragments.

6. The method of any one of claims 1-5, wherein the ligating step is performed before the capturing step.

7. The method of any one of claims 1-5, wherein the capturing step is performed before the ligating step.

8. The method of any one of claims 1-7, wherein the patient sample comprises tissue, urine, cerebral spinal fluid, saliva, feces, ascities, pleural effusion, blood or blood plasma.

9. The method of any one of claims 1-7, wherein the patient sample comprises cell- free nucleic acids in blood plasma.

10. The method of any one of claims 1-9, wherein the hybrid capture probes are at least 30bp in length.

11. The method of claim 10, wherein the hybrid capture probes are between 60bp and 150bp in length, preferably between 80bp and 120bp in length, and further preferably about 100bp in length.

12. The method of any one of claims 1-11, wherein the hybrid capture probes hybridize to at least 30bp, preferably 50bp, more preferably 100bp of the V gene segment and/or J gene segment.

13. The method of any one of claims 1-12, wherein the hybrid capture probes hybridize to at least a portion of the V gene segment and/or J gene segment at either the 3' end or the 5' end of the V gene segment and/or J gene segment respectively.

14. The method of any one of claims 1-13, wherein the screening probes hybridize to at least a portion of the V gene segment.

15. The method of any one of claims 1-13, wherein the screening probes hybridize to at least a portion of the V gene segment at the 3' end.

16. The method of any one of claims 1-15, wherein hybridizing comprises hybridizing under stringent conditions, preferably very stringent conditions.

17. The method of any one of claims 1-16, wherein the collection of nucleic acid hybrid capture probes comprise at least 2, 5, 10, 20, 30, 80, 100, 300, 400, 500, 600, 700, 800 or 900 unique hybrid capture probes.

18. The method of any one of claims 1-16, wherein the collection of nucleic acid hybrid capture probes is sufficient to capture at least 50%, 60%, 70%, 80%, 90% or 99% of known T-Cell receptor and/or immunoglobulin loci clonotypes.

19. The method of any one of claims 1-18, wherein the hybrid capture probes are immobilized on an array.

20. The method of any one of claims 1-18, wherein the hybrid capture probes comprise a label.

21. The method of claim 20, wherein the label is used to distinguish between sequences bound to the screening probes and unbound double stranded fragments.

22. The method of any one of claims 1-21 , wherein the adapter is designed to permit sequencing of the DNA fragment and/or barcoding of the DNA fragment.

23. The method of any one of claims 1-22, wherein preparing the DNA fragments comprises extracting RNA from the patient sample and preparing corresponding cDNA.

24. The method of any one of claims 1-23; further comprising a depletion step, comprising depleting the DNA fragments of non-rearranged sequences using probes that recognize nucleic acid sequences adjacent to V and/or J gene segments in the genome.

25. The method of claim 24, wherein the capturing of DNA fragments using V gene segment and J gene segment hybrid capture probes is performed in separate steps, and in any order with the depletion step, preferably in the following order: J gene capture, depletion, then V gene capture.

26. A method of immunologically classifying a population of T-Cell receptor and/or immunoglobulin sequences, the method comprising:

(a) identifying all sequences containing a V gene segment from the sequences of the DNA fragments by aligning the sequences of the DNA fragments to a library of known V gene segment sequences;

(b) trimming the identified sequences in (a) to remove any sequences corresponding to V gene segments to produce a collection of V-trimmed nucleotide sequences;

(c) identifying all sequences containing a J gene segment in the population of V-trimmed nucleotide sequences by aligning the V-trimmed nucleotide sequences to a library of known J gene segment sequences;

(d) trimming the V-trimmed nucleotide sequences identified in (c) to remove any sequences corresponding to J gene segments to produce VJ-trimmed nucleotide sequences;

(e) identifying any D gene segment comprised in the VJ-trimmed nucleotide sequences identified in (d) by aligning the VJ-trimmed nucleotide sequences to a library of known D gene segment sequences;

(f) for each VJ-trimmed nucleotides sequence identified in (d), assembling a nucleotide sequence comprising the V gene segment, any D gene segment, and the J gene segment identified in steps (a), (e) and (c) respectively;

(g) selecting from the nucleotide sequence assembled in step (f) a junction nucleotide sequence comprising at least the junction between the V gene segment and the J gene segment, including any D gene segment, the junction nucleotide sequence comprising between 18bp and 140bp, preferably 40-100bp, further preferably about 80bp;

and optionally (h) and (i):

(h) translating each reading frame of the junction nucleotide sequence and its complementary strand to produce 6 translated sequences; and

(i) comparing the 6 translated sequences to a library of known CDR3 regions of T- Cell receptor and/or immunoglobulin sequences to identify the CDR3 region in the DNA fragments.

27. The method of claim 26, further comprising, prior to step (a), aligning left and right reads of overlapping initial DNA fragments to produce the DNA fragments on which step (a) is performed.

28. The method of claim 26 or 27, wherein steps (a), (c), (e) are performed with BLASTn and step (i) is performed using expression pattern matching to known sequences and IMGT annotated data.

29. A method of identifying CDR3 regions in T-Cell receptor and/or immunoglobulin sequences, the method comprising:

(a) identifying a V gene segment comprised in the immunoglobulin sequence by aligning the immunoglobulin sequence to a library of known V gene segment sequences;

(b) identifying a J gene segment comprised in the immunoglobulin sequence by aligning the immunoglobulin sequence to a library of known J gene segment sequences;

(c) if V and J gene segments are identified, then comparing the immunoglobulin sequence to a library of known CDR3 regions of T-Cell receptor and/or immunoglobulin sequences to identify any CDR3 region in the immunoglobulin sequence.

30. The method of claim 29, wherein steps (a) and (b) are performed using the Burrows-Wheeler Alignment or other sequence alignment algorithm.

31. The method of claim 29 or 30, wherein if a CDR3 region is identified in step (c), then the method further comprises determining whether the identified V and J gene segments could be rearranged in the same locus using a heuristic approach.

32. The method of claim 29 or 30, wherein if a CDR3 region is not identified in step (c), then the method further comprises determining if a combination of V(D)J gene segments is present based on Smith Waterman Alignment scores.

33. A method for characterizing the immune repertoire of a subject, the immune repertoire comprising the subject's T-Cell population, the method comprising the method of any one of claims 1-25, followed by the method of any one of claims 26- 32.

34. The method of any one of claims 1 -25, for capturing a population of T-Cell receptor sequences.

35. The method of any one of claims 26-28, for immunologically classifying a population of T-Cell receptor sequences.

36. The method of any one of claims 29-32 for identifying CDR3 regions in T-Cell receptor.

37. The method of any one of claims 34-36, for characterizing T-cell clonality for a disease in the subject.

38. The method of claim 37, wherein the subject has cancer.

39. The method of claim 38, wherein the subject has a T-cell related disease, preferably a T-cell lymphoma.

40. The method of any one of claims 33-39, wherein the T-Cell receptor sequences are from tumour infiltrating lymphocytes.

41. The method of claim 40, for identifying therapeutic tumour infiltrating lymphocytes for the purposes of expansion and reinfusion into a patient and/or adoptive cell transfer immunotherapy.

42. The method of any one of claims 33-39, for monitoring T-cell populations/turnover in a subject, preferably a subject with cancer during cancer therapy, preferably immunotherapy.

43. A method for characterizing the immune repertoire of a subject, the immune repertoire comprising the subject's B-Cell population, the method comprising the method of any one of claims 1-25, followed by the method of any one of claims 26- 32.

44. A method of capturing a population of B-Cell receptor sequences with variable regions within a patient sample, said method comprising the methods of any one of claims 1-25.

45. A method of immunologically classifying a population of B-Cell receptor sequences, said method comprising the method of any one of claims 26-28.

46. A method of identifying CDR3 regions in B-Cell receptor sequences comprising the method of any one of claims 29-32.

47. The method of any one of claims 43-46, for characterizing B-cell clonality as a feature of disease in the subject.

48. The method of claim 47, wherein the subject has cancer.

49. The method of claim 48, wherein the subject has a B-cell related disease, plasma cell disorder, preferably a B-cell lymphoma.

50. The method of any one of claims 43-46, for identifying therapeutic B-cells for the purposes of expansion and reinfusion into a patient.

51. The method of any one of claims 43-46, for monitoring B-cell populations/turnover in a subject, preferably a subject with cancer during cancer therapy, preferably immunotherapy.

52. The method of any one of claims 1-32 for detecting minimal residual disease, whereby TCR or immunoglobulin rearrangements may be used as a marker of disease.

53. A library of probes comprising the depletion probes in Table D or at least one of the V-gene and J-gene probes set forth in any of Tables 2.1 , 4, B1 , or B2.