APPLICATION OF A PCR SEQUENCING METHOD, BASED ON DNA BARCODING TECHNIQUE AND DNA INCOMPLETE SHEARING STRATEGY, IN HLA GENOTYPING

• US 20130237432A1
• Filed: 06/30/2011
• Published: 09/12/2013
• Est. Priority Date: 06/30/2010
• Status: Active Grant

• Current Assignee: BGI Genomics Co., Ltd.
• Original Assignee: BGI Genomics Co., Ltd.
• Inventors: Li, Jian, Liu, Ying, Chen, Shiping, Zhang, Xiandong, Zhang, Calfen, Liu, Tao, Zhao, Meiru
• Granted Patent: US 9,957,564 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 whilst 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

1-50. -50. (canceled)

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

1) providing n samples, wherein n is an integer of ≧

1, the samples are from mammalian;

the n samples to be analyzed are divided into m groups, m is an integer and n≧

m≧

1;

2) amplifying;

a pair or multiple pairs of index primers are used for each sample, when there are templates from the sample, PCR amplification is performed under conditions suitable for amplifying the nucleic acid of interest, wherein each pair of index primers consist of a forward index primer and a reverse index primer (both of which may be degenerate primers) comprising primer indexes, wherein the primer indexes comprised in the forward index primer and reverse index primer may be identical or different;

the primer indexes in the pairs of index primers used for different samples are different;

3) pooling;

when n>

1, pooling PCR products from each of the samples together;

4) shearing;

subjecting the amplified products to incomplete shearing, and purifying and recovering;

5) sequencing;

subjecting the recovered DNA mixture to sequencing by using the second generation sequencing technique, Paired-End technique, to obtain sequences of the sheared DNA; and

6) assembling;

corresponding the obtained sequencing data to samples one by one based on the unique primer index for each sample, aligning each sequence read to the DNA reference sequence corresponding to the PCR products by using alignment program, assembling a complete sequence of the nucleic acid of interest from the sequences of the sheared DNA by virtue of sequence overlapping and linkage relationship;

said method has one or more features selected from the group consisting of the following;

a) wherein each pair of primer indexes and a pair of PCR primers form a pair of index primers, wherein forward and reverse PCR primers have a forward primer index and a reverse primer index at 5′

end (or optionally linked by a linker sequence), respectively;

b) wherein said PCR primers are PCR primers for amplification of HLA gene, particularly PCR primers for amplification of HLA-A/B gene, said PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B and Exon 2 of HLA-DRB1, said PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or said PCR primers for amplification of Exon 2 of HLA-DRB1 as shown in Table 7;

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

said PCR primers are as shown in Table 3 or Table 4;

d) wherein said PCR primers are PCR primers for amplification of HLA gene, particularly PCR primers for amplification of HLA-DQB1 gene, said PCR primers for amplification of Exon 2 and/3 of HLA-DQB1 gene;

said PCR primers are as shown in Table 5;

e) wherein said primer indexes are designed for PCR primers, particularly for PCR primers for amplification of a specific gene of HLA, more particularly for PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B and Exon 2 of HLA-DRB1, particularly for PCR primers as shown in Table 1, Table 2 or Table 7;

said primer indexes particularly comprise at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or 95 pairs of the 95 pairs of primer indexes as shown in Table 6 (or the set of primer indexes consisting of 10-95 pairs (for example, 10-95 pairs, 20-95 pairs, 30-95 pairs, 40-95 pairs, 50-95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs) of the 95 pairs of primer indexes as shown in Table

     6); and

the set of index primers comprises at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 to PI-40, or PI-41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, or PI-91 to PI-95 of the 95 pairs of primer indexes as shown in Table 6, or combinations of any two or more of them;

f) wherein said DNA shearing includes chemical shearing methods and physical shearing methods, wherein the chemical shearing methods include enzymatic digestion, and the physical shearing methods include ultrasonic shearing methods or mechanical shearing methods;

g) wherein after said DNA shearing, all the DNA bands between the maximum read length of the sequencer and the applicable maximum DNA length of the sequencer are purified and recovered, wherein said purification and recovery methods include, but are not limited to, recovery by electrophoresis and gel slicing, and recovery by magnetic beads; and

h) wherein said method comprises, in addition to steps

     1) to

     4) as described above, the following steps;

5) constructing a library;

constructing a PCR-free sequencing library by using the library of the sheared PCR products, wherein different library adapters may be added to distinguish different PCR-Free sequencing libraries, all the DNA bands between the maximum read length of the sequencer and the applicable maximum DNA length of the sequencer, particularly DNA fragments of 450 to 750 bp, are purified and recovered;

6) sequencing;

subjecting the recovered DNA mixture to sequencing by using the second generation sequencing technique, particularly Paired-End technique, obtaining the sequences of the sheared DNAs;

7) assembling;

corresponding the obtained sequencing data to the samples one by one based on different library adapter sequences of the libraries and the unique primer index for each sample, aligning each sequence read to the DNA reference sequence corresponding to the PCR products by using alignment program, assembling a complete sequence of the nucleic acid of interest from the sequences of the sheared DNA based on sequence overlapping and linkage relationship.

59. The method as in claim 51, where the sample is from human.

60. The method as in claim 51, where the sample is from human blood.

61. The method as in claim 59, wherein each pair of primer indexes and a pair of PCR primers form a pair of index primers, wherein forward and reverse PCR primers have a forward primer index and a reverse primer index at 5′

end (or optionally linked by a linker sequence), respectively.

62. The method as in claim 59, wherein said PCR primers are PCR primers for amplification of HLA gene, particularly PCR primers for amplification of HLA-A/B gene, said PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B and Exon 2 of HLA-DRB1, said PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or said PCR primers for amplification of Exon 2 of HLA-DRB1 as shown in Table 7.

63. The method as in claim 59, wherein said PCR primers are PCR primers for amplification of HLA gene, particularly PCR primers for amplification of HLA-C gene, said PCR primers for amplification of Exons 2, 3 and/or 4 of HLA-C;

said PCR primers are as shown in Table 3 or Table 4.

64. The method as in claim 59, wherein said PCR primers are PCR primers for amplification of HLA gene, particularly PCR primers for amplification of HLA-DQB1 gene, said PCR primers for amplification of Exon 2 and/3 of HLA-DQB1 gene;

said PCR primers are as shown in Table 5.

65. The method as in claim 59, wherein said primer indexes are designed for PCR primers, particularly for PCR primers for amplification of a specific gene of HLA, more particularly for PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B and Exon 2 of HLA-DRB1, particularly for PCR primers as shown in Table 1, Table 2 or Table 7;

said primer indexes particularly comprise at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or 95 pairs of the 95 pairs of primer indexes as shown in Table 6 (or the set of primer indexes consisting of 10-95 pairs (for example, 10-95 pairs, 20-95 pairs, 30-95 pairs, 40-95 pairs, 50-95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs) of the 95 pairs of primer indexes as shown in Table

     6).

66. The method as in claim 59, wherein said DNA shearing includes chemical shearing methods and physical shearing methods, wherein the chemical shearing methods include enzymatic digestion, and the physical shearing methods include ultrasonic shearing methods or mechanical shearing methods.

67. The method as in claim 59, wherein after said DNA shearing, all the DNA bands between the maximum read length of the sequencer and the applicable maximum DNA length of the sequencer are purified and recovered, wherein said purification and recovery methods include, but are not limited to, recovery by electrophoresis and gel slicing, and recovery by magnetic beads.

68. The method as in claim 59, wherein said method comprises, in addition to steps 1) to 4) as described above, the following steps:

5) constructing a library;

constructing a PCR-free sequencing library by using the library of the sheared PCR products, wherein different library adapters may be added to distinguish different PCR-Free sequencing libraries, all the DNA bands between the maximum read length of the sequencer and the applicable maximum DNA length of the sequencer, particularly DNA fragments of 450 to 750 bp, are purified and recovered;

6) sequencing;

subjecting the recovered DNA mixture to sequencing by using the second generation sequencing technique, particularly Paired-End technique, obtaining the sequences of the sheared DNAs;

7) assembling;

corresponding the obtained sequencing data to the samples one by one based on different library adapter sequences of the libraries and the unique primer index for each sample, aligning each sequence read to the DNA reference sequence corresponding to the PCR products by using alignment program, assembling a complete sequence of the nucleic acid of interest from the sequences of the sheared DNA based on sequence overlapping and linkage relationship.

52. A set of primer indexes, comprising at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or 95 pairs of the 95 pairs of primer indexes as shown in Table 6 (or said set of primer indexes consisting of 10-95 pairs (for example, 10-95 pairs, 20-95 pairs, 30-95 pairs, 40-95 pairs, 50-95 pairs, 60-95 pairs, 70-95 pairs, 80-95 pairs, 90-95 pairs, or 95 pairs) of the 95 pairs of primer indexes as shown in Table 6), and

said set of index primers comprises at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 to PI-40, or PI-41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, or PI-91 to PI-95 of the 95 pairs of primer indexes as shown in Table 6, or combinations of any two or more of them.

53. A set of index primers comprising the set of primer indexes of claim 52 and a pair of PCR primers for amplification of a sequence of interest to be tested, wherein a pair of index primers comprises a pair of primer indexes and a pair of PCR primers, the forward and reverse PCR primer have a forward and a reverse primer index at 5′

end (or optionally linked by a linker sequence), respectively

wherein said PCR primers are PCR primers for amplification of a specific gene of HLA, said PCR primers for amplification of Exons 2, 3, 4 of HLA-A/B gene and Exon 2 of HLA-DRB1, said PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or said PCR primers for amplification of Exon 2 of HLA-DRB1 as shown in Table 7;

said PCR primers for amplification of Exons 2, 3 and/or 4 of HLA-C, said PCR primers are as shown in Table 3 or Table 4;

or said PCR primers for amplification of Exon 2 and/or 3 of HLA-DQB1, said PCR primers are as shown in Table 5.

58. A kit for HLA genotyping or PCR sequencing, comprising the set of index primers of claim 53.

55. PCR primers for HLA genotyping, characterized by that said PCR primers are PCR primers for amplification of Exons 2, 3, 4 of HLA-A/B gene and Exon 2 of HLA-DRB1, said PCR primers for amplification of Exons 2, 3 and 4 of HLA-A/B as shown in Table 1 or Table 2, or PCR primers for amplification of Exon 2 of HLA-DRB1 as shown in Table 7;

said PCR primers for amplification of Exons 2, 3 and/or 4 of HLA-C, said PCR primers are as shown in Table 3 or Table 4;

or PCR primers for amplification of Exons 2 and/or 3 of HLA-DQB1, said PCR primers are as shown in Table 5.

56. A sequencing method using the PCR primers of claim 55, comprising

providing a sample, particularly a blood sample, said blood sample is from mammalian, particularly human;

amplifying;

amplifying DNA from the blood sample with the PCR primers to obtain PCR products, and purifying the PCR products;

sequencing;

subjecting the PCR products to sequencing, the sequencing method may be Sanger sequencing method, or the second generation sequencing method.

57. A kit for HLA genotyping, comprising the PCR primers of claim 55.