A Bead-based Normalization for Uniform Sequencing depth (BeNUS) protocol for multi-samples sequencing exemplified by HLA-B

BMC Genomics

Ituro Inoue²  Hideki Nagasaki¹  Shigeki Mitsunaga³  Kazuyoshi Hosomichi² 

[1] Genome Informatics Laboratory, Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, 1111 Yata, Mishima, 411-8540 Shizuoka, Japan;Division of Human Genetics, National Institute of Genetics, 1111 Yata, Mishima, 411-8540 Shizuoka, Japan;Department of Molecular Life Sciences, Tokai University School of Medicine, 143 Shimokasuya, 259-1143 Isehara, Kanagawa, Japan

关键词: Next-generation sequencing;    HLA;   

Others  :  1216341
DOI  :  10.1186/1471-2164-15-645

 received in 2014-01-28, accepted in 2014-07-30,  发布年份 2014

【 摘 要 】

Background

Human leukocyte antigen (HLA) is a group of genes that are extremely polymorphic among individuals and populations and have been associated with more than 100 different diseases and adverse drug effects. HLA typing is accordingly an important tool in clinical application, medical research, and population genetics. We have previously developed a phase-defined HLA gene sequencing method using MiSeq sequencing.

Results

Here we report a simple, high-throughput, and cost-effective sequencing method that includes normalized library preparation and adjustment of DNA molar concentration. We applied long-range PCR to amplify HLA-B for 96 samples followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. After sequencing, we observed low variation in read percentages (0.2% to 1.55%) among the 96 demultiplexed samples. On this basis, all the samples were amenable to haplotype phasing using our phase-defined sequencing method. In our study, a sequencing depth of 800x was necessary and sufficient to achieve full phasing of HLA-B alleles with reliable assignment of the allelic sequence to the 8 digit level.

Conclusions

Our HLA sequencing method optimized for 96 multiplexing samples is highly time effective and cost effective and is especially suitable for automated multi-sample library preparation and sequencing.

【 授权许可】

   
2014 Hosomichi et al.; licensee BioMed Central Ltd.

【 图 表 】

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