HLA遺伝⼦完全配列決定
パイプラインの構築
国⽴遺伝学研究所 総合遺伝研究系
人類遺伝研究部門
細道一善
2014年12月26日(⾦)
平成26年度 統合データ解析トライアル 中間報告会©2014 細道一善(国立遺伝学研究所) licensed under CC表示2.1日本

研究開発内容
1. 研究開発の必要性
2. 研究項目と現在の状況-haplotype phasingプログラムの開発-解析手法のパイプライン化

HLA領域のゲノム配列
6p21

Shiina T et al. 2009

HLA領域の特徴
252の遺伝⼦、6つの古典的HLA遺伝⼦と少なくとも132のタンパク質をコードする遺伝⼦を含む
極めて⾼度な多型性を示す
移植のみならず100以上の疾患と関連する

HLA クラスI α1およびα2ドメインの多様性
5 amino acids

33

4333

peptideα2α3α1

日本人で頻度の⾼いHLA-B アレル5種間β2Μアミノ酸配列での類似性: 88.9%
塩基配列での類似性: 93.9%

TMCY

HLA class I

IMGT/HLAデータベースに登録されているHLAアレル数
Numbers of HLA Alleles
HLA Class I Alleles

8,976

HLA Class II Alleles

2,870

HLA Alleles

11,846

HLA Class IGeneABC

Alleles

2,884

3,589

2,375

Proteins

2,041

2,668

1,677

133

11971

Nulls
HLA Class II
GeneDRB

DQB1

DPB1

Alleles

1,642

664422

Proteins

1,211

435

351

371610Nulls

IMGT/HLA database Version Report - 3.17 (2014-07)

IMGT/HLAデータベースに登録されている
HLAアレル数

HLAアレルの命名法

HLAと薬剤副作用との関連性
薬剤副作用関連を示すHLA型

陽性率(%)

オッズ比Tiopronin(重⾦属)と肝内胆汁うっ滞

HLA-A*33:039341.5

Carbamazepine(抗痙攣剤)とStevens-Johnson 症候群

HLA-B*15:02

100895.5

Abacavir(抗HIV剤)と胃腸障害、嗜眠、低血圧による致死副作用

HLA-B*57:01

78117.5

Allopurinol(抗痛風、抗尿酸血症剤)と薬疹

HLA-B*58:01

100393.5

Ticlopidine(抗血小板剤)と肝障害

HLA-A*33:03

8636.5

Amoxicillin-clavulanate potassium(抗生物質)と肝障害

HLA-DRB1*15:01

5735.6

Flucloxacillin(抗生物質)と肝障害

HLA-B*57:01

8480.6

原因がHLA遺伝⼦そのものであるか、連鎖不平衡によりHLA遺伝⼦と関連しているようにみえるかはAbacavirを除き不明

HLAタイピングによる予防的診断
HLA-B*57:01
positive

HLA-B*57:01
negative

ADR negative25794

ADR positive230ADR
Positive %48%0%Mallal S et al. N Engl J Med. 2008

研究開発内容
1. 研究開発の必要性
2. 研究項目と現在の状況-haplotype phasingプログラムの開発-解析手法のパイプライン化

対象データおよび対象データベース

HLA database 登録配列

MHC Haplotype Consortium WebResource

Workflow of phase-defined sequencing
Paired-read fastq

hg19 reference
Alignment to HLA gene sequence of hg19 reference
BWA, Samtools, Picard

bam file

Detect SNVs and Indels
Haplotypecaller in GATK

vcf file

Phase haplotype using heterozygous SNVs on PE reads
Perl script

SNV list

Divide alignment into two phased one as haplotype
Perl script

2 sam files

Create consensus sequence
FastaAlternateReferenceMaker in GATK, Perl script

fasta file

Haplotype sequenceセルラインにおけるHLA-BのNGSデータ
COX
B*07:02:01

PGF
B*08:01:01

HLA-B

セルラインにおけるHLA-BのNGSデータ
COX
B*07:02:01

PGF
B*08:01:01

COX
B*07:02:01+PGF
B*08:01:01

HLA-B

Workflow of phase-defined sequencing
Paired-read fastq

hg19 reference
Alignment to HLA gene sequence of hg19 reference
BWA, Samtools, Picard

bam file

Detect SNVs and Indels
Haplotypecaller in GATK

vcf file

Phase haplotype using heterozygous SNVs on PE reads
Perl script

SNV list

Divide alignment into two phased one as haplotype
Perl script

2 sam files

Create consensus sequence
FastaAlternateReferenceMaker in GATK, Perl script

fasta file

Haplotype sequenceHLA遺伝⼦配列完全決定のワークフロー
ex7 ex6

ex5 ex4

HLA-B

ex3

ex2

ex1

Before phasing

Phased haplotype 1

B*51:01:01

Phased haplotype 2

B*15:02:01

実データ解析へのパイプライン最適化
Paired-read fastq

hg19 reference

Alignment

Alignment to HLA gene sequence of hg19 reference
Detect SNVs and Indels
YesHeterozygous?

NoComplete haplotype sequence

Phase haplotype using heterozygous SNVs on PE reads
Divide alignment into two phased one as haplotype
Phased completely?
Yes

No

Unphased heterozygous SNVs?

NoYes
Create alignment for only homozygous region
Merge two alignments
Cleaning of phased alignment
Complete haplotype sequence

Partial haplotype sequence実データ解析へのパイプライン最適化
Paired-read fastq

hg19 reference

Alignment

Alignment to HLA gene sequence of hg19 reference
Detect SNVs and Indels
Yes

Heterozygous?

No

Complete haplotype sequence

Phase haplotype using heterozygous SNVs on PE reads
Divide alignment into two phased one as haplotype
Phased completely?
Yes

No

Unphased heterozygous SNVs?

NoYes
Create alignment for only homozygous region
Merge two alignments
Cleaning of phased alignment
Complete haplotype sequence

Partial haplotype sequenceAlignment algorithms and mapped rate

Average
MinMax

BWA-backtrack(max diff = 80% )BWA-MEM

94.7%
88.8%
99.4%

99.6%
99.1%
99.9%

BWA-backtrack : illumina short reads
BWA-MEM : short reads and long reads (up to 1 Mb)

実データ解析へのパイプライン最適化
Paired-read fastq

hg19 reference

Alignment

Alignment to HLA gene sequence of hg19 reference
Detect SNVs and Indels
Yes

Heterozygous?

No

Complete haplotype sequence

Phase haplotype using heterozygous SNVs on PE reads
Divide alignment into two phased one as haplotype
Phased completely?
Yes

No

Unphased heterozygous SNVs?

No
Yes
Create alignment for only homozygous region
Merge two alignments
Cleaning of phased alignment
Complete haplotype sequence

Partial haplotype sequenceAlignment view of heterozygous samples
that had few SNVs or one SNV in one exon
ex20785
4 SNVs in exon21018
7 SNVs in exon21117
3 SNVs in exon21175
3 SNVs in exon21224
3 SNVs in exon30979
1 SNV in exon5ex3ex5

Partial phasing for only particular exon

0785
diplotype

haplotype1

haplotype2

Alignment of homozygous region
0785
diplotype

Homozygous
regionMerging alignments of homozygous
and heterozygous region
Homozygous
regionHeterozygous
regionHaplotype1

Mergedalignment
Haplotype1

Merging alignments of homozygous
and heterozygous region
Homozygous
regionHeterozygous
regionHaplotype2

Mergedalignment
Haplotype2

Partial phasing for only particular exon

0785
diplotype

haplotype1haplotype2

Alignment of HLA-B*52:01

0605
52:01
homozygous
0616

0663

0785

1011

実データ解析へのパイプライン最適化
Paired-read fastq

hg19 reference

Alignment

Alignment to HLA gene sequence of hg19 reference
Detect SNVs and Indels
Yes

Heterozygous?

No

Complete haplotype sequence

Phase haplotype using heterozygous SNVs on PE reads
Divide alignment into two phased one as haplotype
Phased completely?
Yes

No

Unphased heterozygous SNVs?

No
Yes
Create alignment for only homozygous region
Merge two alignments
Cleaning of phased alignment
Complete haplotype sequence

Partial haplotype sequence

PCR in library preparation andsequencing
Nextera kit
SampleInput

Tagmentation

PCR

Beads normalization

MiSeq
Cluster generation

Sequencing

DNA recombination during PCR

Yu et al. Biotechniques. 2006

DNA recombination during PCR

Average recombination frequency
RFLP: 35.7% ± 5.0%cloning: 26.4% (91 clones)
Yu et al. Biotechniques. 2006

Artifact SNVs caused by recombination

afterbefore

Removing recombinant sequence reads(SNVs)

afterbefore

Removing recombinant sequence reads(Indel)

afterbefore

Alignment of HLA-B*40:06実データ解析へのパイプライン最適化
Paired-read fastq

hg19 reference

Alignment

Alignment to HLA gene sequence of hg19 reference
Detect SNVs and Indels
Yes

Heterozygous?

No

Complete haplotype sequence

Phase haplotype using heterozygous SNVs on PE reads
Divide alignment into two phased one as haplotype
Phased completely?
Yes

No

Unphased heterozygous SNVs?

No
Yes
Create alignment for only homozygous region
Merge two alignments
Cleaning of phased alignment
Complete haplotype sequence

Partial haplotype sequence