HIGH RESOLUTION, HIGH THROUGHPUT HLA GENOTYPING BY CLONAL SEQUENCING

• US 20100086914A1
• Filed: 10/03/2008
• Published: 04/08/2010
• Est. Priority Date: 10/03/2008
• Status: Abandoned Application

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: BENTLEY, Gordon, Erlich, Henry, Higuchi, Russell
• Application Number:
  US12/245,666
• Publication Number:
  US 20100086914A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention provides methods and reagent for performing full, multi-locus HLA genotyping for multiple individuals in a single sequencing run using clonal sequencing.

Claims

1. A method of determining the HLA genotypes for the HLA genes HLA-A, HLA-B, HLA-C, DRB1, DQA1, DQB1, DPA1, and DPB1 for more than one individuals in parallel, the method comprising:

(a) for each individual, amplifying the exons of the HLA-A, HLA-B, HLA-C, DRB1, DQA1, DQB1, DPA1, and DPB1 genes that comprises polymorphic sites to obtain HLA-A, HLA-B, HLA-C, DRB1, DQA1, DQB1, DPA1, and DPB1 amplicons for each individual, wherein each amplification reaction is performed with a forward primer and a reverse primer to amplify an HLA gene exon, where;

(i) the forward primer comprises the following sequences, from 5′

to 3″

;

an adapter sequence, a molecular identification sequence, and an HLA-hybridizing sequence; and

(ii) the reverse primer comprises the following sequences, from 5′

to 3′

;

an adapter sequence, a molecular identification sequence, and an HLA-hybridizing sequence;

(b) pooling HLA amplicons from more than one individual and performing emulsion PCR;

(c) determining the sequence of the HLA-A, HLA-B, HLA-C, DRB1, DQA1, DQB1, DPA1, and DPB1 amplicon for each individual using pyrosequencing in parallel; and

(d) assigning the HLA alleles to each individual by comparing the sequence of the HLA amplicons to known HLA sequences to determine which HLA alleles are present in the individual.

2. The method of claim 1, wherein the forward primer for obtaining an HLA amplicon has the sequence of an HLA-binding region of a primer set forth in Table 1.

3. The method of claim 2, wherein the forward primer has an adapter region of a primer set forth in Table 1.

4. The method of claim 3, wherein the forward primer has an individual identification tag of a primer set forth in Table 1.

5. The method of claim 4, wherein the forward primer has a sequence of a primer set forth in Table 1.

6. The method of claim 1, wherein the reverse primer for obtaining an HLA amplicon has the sequence of an HLA-binding region of a primer set forth in Table 1.

7. The method of claim 6, wherein the reverse primer has an adapter region of a primer set forth in Table 1.

8. The method of claim 7, wherein the reverse primer has an individual identification tag of a primer set forth in Table 1.

9. The method of claim 8, wherein the reverse primer has a sequence of a primer set forth in Table 1.

10. The method of claim 1, wherein the forward primer for obtaining an HLA amplicon has the sequence of an HLA-hybridizing region of a primer set forth in Table 1;

and the reverse primer for obtaining the HLA amplicon has the sequence of an HLA-hybridizing region of a primer set forth in Table 1.

11. The method of claim 10, wherein the forward primer has an adapter region of a primer set forth in Table 1;

and the reverse primer has an adapter region of a primer set forth in Table 1.

12. The method of claim 1, wherein the forward primer has an individual identification tag of a primer set forth in Table 1 and the reverse primer has an individual identification tag of a primer set forth in Table 1.

13. The method of claim 12, wherein the forward primer has a sequence of a primer set forth in Table 1 and the reverse primer has a sequence of a primer set forth in Table 1.

14. A kit comprising primer pairs for obtaining HLA amplicons f to determine the HLA genotypes for the HLA genes HLA-A, HLA-B, HLA-C, DRB1, DQA1, DQB1, DPA1, and DPB1 for more than one individuals in parallel, wherein the primer pairs comprise a forward primer and a reverse primer to amplify an HLA gene exon, where:

(i) the forward primer comprises the following sequences, from 5′

to 3″

;

an adapter sequence, a molecular identification sequence, and an HLA sequence; and

(ii) the reverse primer comprises the following sequences, from 5′

to 3′

;

an adapter sequence, a molecular identification sequence, and an HLA sequence.

15. The kit of claim 14, wherein the primer pairs comprise forward and reverse primers set forth in Table 1.

16. A kit comprising one or more primer pairs, wherein each primer pair comprises a forward primer for obtaining an HLA amplicon that has the sequence of an HLA-binding region of a primer set forth in Table 1;

and a reverse primer for obtaining the HLA amplicon that has the sequence of an HLA-binding region of a primer set forth in Table 1.

17. The kit of claim 16, wherein the forward primer has an adapter region of a primer set forth in Table 1;

and the reverse primer has an adapter region of a primer set forth in Table 1.

18. The kit of claim 17, wherein the forward primer has an individual identification tag of a primer set forth in Table 1 and the reverse primer has an individual identification tag of a primer set forth in Table 1.

19. The kit of claim 18, wherein the forward primer has a sequence of a primer set forth in Table 1 and the reverse primer has a sequence of a primer set forth in Table 1.

20. The kit of claim 16, wherein the kit comprises fifteen HLA primer pairs, where the primer pairs amplify exon 2, exon 3, and exon 4 of HLA-A;

exon 2, exon 3, and exon 4 of HLA-B;

exon 2, exon 3, and exon 4 of HLA-C;

exon 2 of DRB1, exon 2 of DPB1, exon 2 of DPA1, exon 2 of DQA1; and

exon 2 and exon 3 of DQB1.