Application of a PCR sequencing method, based on DNA barcoding technique and DNA incomplete shearing strategy, in HLA genotyping

• US 9,957,564 B2
• Filed: 06/30/2011
• Issued: 05/01/2018
• Est. Priority Date: 06/30/2010
• Status: Active Grant

• Current Assignee: BGI Genomics Co., Ltd.
• Original Assignee: BGI Genomics Co., Ltd.
• Inventors: Li, Jian, Chen, Shiping, Zhang, Xiandong, Liu, Ying, Zhang, Caifen, Liu, Tao, Zhao, Meiru
• Primary Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US13/807,660
• Publication Number:
  US 20130237432A1
• Time in Patent Office: 2,497 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• 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 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

The invention provides a PCR sequencing method, wherein the combination of primer indexes, DNA incomplete shearing strategy and the second generation sequencing technique (Paired-End sequencing technique) can make the length of PCR products that can be sequenced by a sequencer longer than the maximum sequencing length of the sequencer while making full use of the characteristics of the second generation sequencing technique such as high throughput and low cost, thereby greatly broadening its applicable scope. In addition, the present invention also provides primer indexes for the PCR sequencing method and the use of the method in genotyping, particularly in HLA analysis, and also provides the PCR primers used, particularly the PCR primers for HLA-A, B, HLA-C and HLA-DQB1 gene.

Claims

1. A method for determining the nucleotide sequence of a nucleic acid of interest, comprising:

a) providing n samples, wherein n is an integer of ≥

1;

b) optionally dividing the n samples into m groups, wherein m is an integer and n≥

m≥

1;

c) performing PCR amplification on the samples under conditions suitable for amplifying the nucleic acid of interest when templates from the samples are available, wherein a pair or multiple pairs of index primers are used for each sample, each pair of index primers consists of a forward index primer and a reverse index primer, and each index primer consists of a PCR primer and a primer index added to the 5′

-end of the PCR primer, wherein the primer index of the forward index primer and the primer index of the reverse index primer in each pair of index primers are identical or different, and wherein the primer indexes in the pair of index primers used for different samples are different;

d) pooling products of the PCR amplification from each of the samples together;

e) subjecting the amplified products to incomplete shearing to obtain a mixture of intact un-sheared PCR products and partially sheared PCR products, and purifying and recovering said mixture of intact un-sheared PCR products and partially sheared PCR products;

f) constructing a PCR-free sequencing library based on the mixture of intact un-sheared PCR products and partially sheared PCR products recovered in e), wherein different library adapters are added to distinguish different PCR-Free sequencing libraries;

g) purifying and recovering DNA bands between the maximum read length and the maximum applicable DNA length of a sequencer using the second generation sequencing technique;

h) subjecting the recovered DNA mixture to the sequencer to obtain sequences of the sheared DNA; and

i) matching obtained sequencing data to corresponding samples based on a unique primer index for each sample, aligning obtained sequences of the sheared DNA against DNA reference sequences corresponding to the PCR products, and assembling a complete sequence of the nucleic acid of interest from the obtained sequences of the sheared DNA based on sequence overlap and linkage relationship, whereby a length of the complete sequence of the nucleic acid of interest exceeds a maximum read length of a sequencer used for said sequencing.

2. The method of claim 1, wherein:

a) said PCR primers are PCR primers for amplification of HLA gene, HLA-A/B gene, Exons 2, 3 and 4 of HLA-A/B, or Exon 2 of HLA-DRB1;

b) said PCR primers are PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B as shown in SEQ ID NO;

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12;

c) said PCR primers are PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B as shown in SEQ ID NO;

13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24;

d) said PCR primers are PCR primers for amplification of Exon 2 of HLA-DRB1 as shown in SEQ ID NO;

231, 232, 233, 234, 235, 236, 237 and 238;

e) said PCR primers are PCR primers for amplification of HLA gene, HLA-C gene, or Exons 2, 3 and/or 4 of HLA-C;

f) said PCR primers are PCR primers for amplification of Exons 2, 3 and/or 4 of HLA-C as shown in SEQ ID NO;

25, 26, 27, 28, 29 and 30;

g) said PCR primers are PCR primers for amplification of Exons 2, 3 and/or 4 of HLA-C as shown in SEQ ID NO;

31, 32, 33, 34, 35 and 36;

h) said PCR primers are PCR primers for amplification of HLA gene, HLA-DQB1 gene, or Exon 2 and/3 of HLA-DQB1 gene;

or

i) said PCR primers are PCR primers for amplification of Exon 2 and/3 of HLA-DQB1 gene as shown in SEQ ID NO;

37, 38, 39 and 40.

3. The method of claim 1, wherein said primer indexes are designed for:

a) PCR primers for amplification of a specific gene of HLA;

b) PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B and Exon 2 of HLA-DRB1;

c) PCR primers shown in SEQ ID NO;

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12;

d) PCR primers shown in SEQ ID NO;

13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24;

or

e) PCR primers shown in SEQ ID NO;

231, 232, 233, 234, 235, 236, 237 and 238.

4. The method of claim 1, wherein at least 10 pairs of primer indexes selected from the group consisting of primer index Nos. PI-1 to PI-95 as set forth in the table below are used

5. The method of claim 1, wherein said DNA shearing is performed by chemical shearing and/or physical shearing.

6. The method of claim 5, wherein said chemical shearing comprises enzymatic digestion.

7. The method of claim 5, wherein said physical shearing comprises ultrasonic shearing or mechanical shearing.

8. The method of claim 1, wherein, in step g), the DNA bands between the maximum read length and the maximum applicable DNA length of the second generation sequencer are purified and recovered by electrophoresis and gel slicing, or by magnetic beads.

9. The method of claim 1, wherein the sample is from human.

10. The method of claim 9, wherein the sample is human blood.

11. The method of claim 1, wherein the sequencer uses Paired-End technique.

12. The method of claim 1, wherein the sequencer is a Hiseq 2000 sequencer, an Illumina GA sequencer, an Illumina Solexa sequencer, or a Roche454 sequencer.

13. The method of claim 1, wherein the DNA bands purified and recovered in step g) have a length ranging from 450 bp to 700 bp, and wherein the sequencer is an Illumina GA sequencer.

14. A method of determining HLA genotype of a sample, comprising:

a) sequencing a sample from a patient by the method of claim 2;

b) aligning the sequencing results against sequence data of Exons of HLA, Exons 2, 3 and 4 of HLA-A/B, Exons 2, 3 and/or Exon 4 of HLA-C, Exon 2 and/or 3 of HLA-DQB1 gene and/or Exon 2 of HLA-DRB1 in HLA database; and

c) determine the HLA genotype of the sample if the result of sequence alignment shows 100% match.

15. The method of claim 14, wherein said primer indexes are designed for:

a) PCR primers for amplification of a specific gene of HLA;

b) PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B and Exon 2 of HLA-DRB1;

c) PCR primers shown in SEQ ID NO;

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12;

d) PCR primers shown in SEQ ID NO;

13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24;

or

e) PCR primers shown in SEQ ID NO;

231, 232, 233, 234, 235, 236, 237 and 238.

16. The method of claim 14, wherein at least 10 pairs of primer indexes selected from the group consisting of primer index Nos. PI-1 to PI-95 as set forth in the table below are used

17. The method of claim 14, wherein said DNA shearing is performed by chemical shearing and/or physical shearing, wherein said chemical shearing comprises enzymatic digestion, and wherein said physical shearing comprises ultrasonic shearing or mechanical shearing.

18. The method of claim 14, wherein the sample is from human.

19. The method of claim 18, wherein the sample is human blood.

20. The method of claim 14, wherein the DNA bands purified and recovered for sequencing have a length ranging from 450 bp to 700 bp, and wherein the second generation sequencer is an Illumina GA sequencer.