PMID- 23714642
OWN - NLM
STAT- MEDLINE
DCOM- 20131030
LR  - 20211021
IS  - 1471-2164 (Electronic)
IS  - 1471-2164 (Linking)
VI  - 14
DP  - 2013 May 28
TI  - Phase-defined complete sequencing of the HLA genes by next-generation sequencing.
PG  - 355
LID - 10.1186/1471-2164-14-355 [doi]
AB  - BACKGROUND: The human leukocyte antigen (HLA) region, the 3.8-Mb segment of the 
      human genome at 6p21, has been associated with more than 100 different diseases, 
      mostly autoimmune diseases. Due to the complex nature of HLA genes, there are 
      difficulties in elucidating complete HLA gene sequences especially HLA gene 
      haplotype structures by the conventional sequencing method. We propose a novel, 
      accurate, and cost-effective method for generating phase-defined complete 
      sequencing of HLA genes by using indexed multiplex next generation sequencing. 
      RESULTS: A total of 33 HLA homozygous samples, 11 HLA heterozygous samples, and 3 
      parents-child families were subjected to phase-defined HLA gene sequencing. We 
      applied long-range PCR to amplify six HLA genes (HLA-A, -C, -B, DRB1, -DQB1, and 
      -DPB1) followed by transposase-based library construction and multiplex 
      sequencing with the MiSeq sequencer. Paired-end reads (2 x 250 bp) derived from 
      the sequencer were aligned to the six HLA gene segments of UCSC hg19 allowing at 
      most 80 bases mismatch. For HLA homozygous samples, the six amplicons of an 
      individual were pooled and simultaneously sequenced and mapped as an 
      individual-tagging method. The paired-end reads were aligned to corresponding 
      genes of UCSC hg19 and unambiguous, continuous sequences were obtained. For HLA 
      heterozygous samples, each amplicon was separately sequenced and mapped as a 
      gene-tagging method. After alignments, we detected informative paired-end reads 
      harboring SNVs on both forward and reverse reads that are used to separate two 
      chromosomes and to generate two phase-defined sequences in an individual. 
      Consequently, we were able to determine the phase-defined HLA gene sequences from 
      promoter to 3'-UTR and assign up to 8-digit HLA allele numbers, regardless of 
      whether the alleles are rare or novel. Parent-child trio-based sequencing 
      validated our sequencing and phasing methods. CONCLUSIONS: Our protocol generated 
      phased-defined sequences of the entire HLA genes, resulting in high resolution 
      HLA typing and new allele detection.
FAU - Hosomichi, Kazuyoshi
AU  - Hosomichi K
AD  - Division of Human Genetics, National Institute of Genetics, Shizuoka, Japan.
FAU - Jinam, Timothy A
AU  - Jinam TA
FAU - Mitsunaga, Shigeki
AU  - Mitsunaga S
FAU - Nakaoka, Hirofumi
AU  - Nakaoka H
FAU - Inoue, Ituro
AU  - Inoue I
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20130528
PL  - England
TA  - BMC Genomics
JT  - BMC genomics
JID - 100965258
RN  - 0 (HLA Antigens)
SB  - IM
MH  - Child
MH  - Female
MH  - HLA Antigens/*genetics
MH  - Heterozygote
MH  - High-Throughput Nucleotide Sequencing/*methods
MH  - Homozygote
MH  - Humans
MH  - Male
MH  - Pedigree
MH  - Sequence Analysis, DNA/*methods
PMC - PMC3671147
EDAT- 2013/05/30 06:00
MHDA- 2013/10/31 06:00
PMCR- 2013/05/28
CRDT- 2013/05/30 06:00
PHST- 2013/01/15 00:00 [received]
PHST- 2013/05/20 00:00 [accepted]
PHST- 2013/05/30 06:00 [entrez]
PHST- 2013/05/30 06:00 [pubmed]
PHST- 2013/10/31 06:00 [medline]
PHST- 2013/05/28 00:00 [pmc-release]
AID - 1471-2164-14-355 [pii]
AID - 10.1186/1471-2164-14-355 [doi]
PST - epublish
SO  - BMC Genomics. 2013 May 28;14:355. doi: 10.1186/1471-2164-14-355.