System and method for detection of HLA Variants

• US 20100261189A1
• Filed: 04/12/2010
• Published: 10/14/2010
• Est. Priority Date: 10/03/2008
• Status: Abandoned Application

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Bentley, Gordon, Holcomb, Cherie, Erlich, Henry A., Higuchi, Russell Gene
• Application Number:
  US12/798,877
• Publication Number:
  US 20100261189A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method for detecting one or more HLA sequence types is described that comprises the steps of: amplifying a plurality of first amplicons from a double stranded nucleic acid sample, wherein the first amplicons are amplified with a plurality of pairs of nucleic acid primers that define exons 2 and 3 of both strands of HLA loci from the group consisting of HLA-A, HLA-B, and HLA-C; amplifying the first amplicons to produce a plurality of populations of second amplicons, wherein each population of second amplicons is clonally amplified from one of the first amplicons; sequencing the plurality of populations of second amplicons to generate a nucleic acid sequence composition for each of the plurality of second amplicons; and detecting variation in the sequence composition from one or more of the second amplicons for one or more of the HLA loci.

Claims

1. A method for detecting one or more HLA sequence variants, comprising the steps of:

(a) amplifying a plurality of first amplicons from a double stranded nucleic acid sample, wherein the first amplicons are amplified with a plurality of pairs of nucleic acid primers that define exons 2 and 3 of both strands of HLA loci selected from the group consisting of HLA-A, HLA-B, and HLA-C;

(b) amplifying the first amplicons to produce a plurality of populations of second amplicons, wherein each population of second amplicons is clonally amplified from one of the first amplicons;

(c) sequencing the plurality of populations of second amplicons to generate a nucleic acid sequence composition for each of the plurality of second amplicons; and

(d) detecting variation in the sequence composition from one or more of the second amplicons for one or more of the HLA loci.

2. The method of claim 1, wherein:

the pairs of nucleic acid primers comprise sequence composition selected from a plurality of primers listed in Tables 4 and 5.

3. The method of claim 1, wherein:

the plurality of pairs of nucleic acid primers define exons 1, 4, and 5 of the HLA loci.

4. The method of claim 3, wherein:

the first amplicons comprise an amplicon comprising sequence composition of exon 1, intron 1, and exon 2 of the HLA loci.

5. The method of claim 3, wherein:

the first amplicons comprise an amplicon comprising sequence composition of exon 3 of the HLA loci.

6. The method of claim 3, wherein:

the first amplicons comprise an amplicon comprising sequence composition of exon 4, intron 4, and exon 5 of the HLA loci.

7. The method of claim 1, wherein:

the plurality of pairs of nucleic acid primers define exons 6, and 7 of the HLA-C locus.

8. The method of claim 7, wherein:

the first amplicons comprise an amplicon comprising sequence composition of exon 6, intron 6, and exon 7 of the HLA-C locus.

9. The method of claim 1, wherein:

the plurality of pairs of nucleic acid primers for the HLA-A locus enable the sequencing of one or more exons in a forward and a reverse direction.

10. The method of claim 1, wherein:

the plurality of pairs of nucleic acid primers for the HLA-B locus enable the sequencing of one or more exons in a forward and a reverse direction.

11. The method of claim 1, wherein:

the plurality of pairs of nucleic acid primers for the HLA-C enable the sequencing of one or more exons in a forward and a reverse direction.

12. The method of claim 1, further comprising:

a plurality of adaptors each comprising an individual primer from the pairs of the nucleic acid primers.

13. The method of claim 12, wherein:

one or more of the plurality of adaptors comprise an MID identifier.

14. The method of claim 13, wherein:

the MID identifier enables pooling of the first amplicons derived from a plurality of the nucleic acid samples, wherein the populations of the second amplicons amplified from the pooled first amplicons are sequenced in parallel.

15. The method of claim 12, wherein:

the plurality of adaptors comprise a general adaptor element and a key element.

16. The method of claim 1, wherein:

each population of second amplicons is immobilized on a bead substrate.

17. The method of claim 1, wherein:

the populations of second amplicons are clonally amplified using an emulsion PCR process.

18. The method of claim 1, wherein:

the plurality of populations of second amplicons are sequenced in parallel.

19. The method of claim 1 further comprising the step of:

(e) associating the variation with an HLA type.

20. The method of claim 19 wherein:

the association of variation and HLA type is known.

38. A kit for detecting one or more HLA sequence variants, comprising:

a plurality of the pairs of nucleic acid primers employed to amplify the first amplicons of claim 1.

21. A method for detecting one or more HLA sequence variants, comprising the steps of:

(a) amplifying a plurality of first amplicons from a double stranded nucleic acid sample, wherein the first amplicons are amplified with a plurality of pairs of nucleic acid primers that define exon 2 of both strands of HLA loci selected from the group consisting of DRB1, DQA1, DQB1, DPA1, DPB1;

(b) amplifying the first amplicons to produce a plurality of populations of second amplicons, wherein each population of second amplicons is clonally amplified from one of the first amplicons;

(c) sequencing the plurality of populations of second amplicons to generate a nucleic acid sequence composition for each of the plurality of second amplicons; and

(d) detecting variation in the sequence composition from one or more of the second amplicons for one or more of the HLA loci.

22. The method of claim 21, wherein:

the pairs of nucleic acid primers comprise sequence composition selected from a plurality of primers listed in Tables 4 and 5.

23. The method of claim 21, wherein:

the plurality of pairs of nucleic acid primers for the DRB1 locus are generic and further enable amplification of loci are selected from the group consisting of DRB3, DRB4, and DRB5 loci.

24. The method of claim 23, wherein:

the plurality of pairs of nucleic acid primers for the DRB1, 3, 4, and 5 loci enable the of the sequencing of exon 2 in a forward and a reverse direction.

25. The method of claim 21, wherein:

the plurality of pairs of nucleic acid primers for the DQA1 locus enable the sequencing of exon 2 in a forward and a reverse direction.

26. The method of claim 21, wherein:

the plurality of pairs of nucleic acid primers for the DQB1 locus enable the sequencing of exon 2 and exon 3 in a forward and a reverse direction.

27. The method of claim 21, wherein:

the plurality of pairs of nucleic acid primers for the DPA1 locus enable the sequencing of exon 2 in a forward and a reverse direction.

28. The method of claim 21, wherein:

the plurality of pairs of nucleic acid primers for the DPB1 locus enable the sequencing of exon 2 in a forward and a reverse direction.

29. The method of claim 21, further comprising:

a plurality of adaptors each comprising an individual primer from the pairs of the nucleic acid primers.

30. The method of claim 29, wherein:

one or more of the plurality of adaptors comprise an MID identifier.

31. The method of claim 30, wherein:

the MID identifier enables pooling of the first amplicons derived from a plurality of the nucleic acid samples, wherein the populations of the second amplicons amplified from the pooled first amplicons are sequenced in parallel.

32. The method of claim 29, wherein:

the plurality of adaptors comprise a general adaptor element and a key element.

33. The method of claim 21, wherein:

each population of second amplicons is immobilized on a bead substrate.

34. The method of claim 21, wherein:

the populations of second amplicons are clonally amplified using an emulsion PCR process.

35. The method of claim 21, wherein:

the plurality of populations of second amplicons are sequenced in parallel.

36. The method of claim 21 further comprising the step of:

(e) associating the variation with an HLA type.

37. The method of claim 36 wherein:

the association of variation and HLA type is known.

39. A kit for detecting one or more HLA sequence variants, comprising:

a plurality of the pairs of nucleic acid primers employed to amplify the first amplicons of claim 21.