A Bead-based Normalization for Uniform Sequencing depth (BeNUS) protocol for multi-samples sequencing exemplified by HLA-B |
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Authors: | Kazuyoshi Hosomichi Shigeki Mitsunaga Hideki Nagasaki Ituro Inoue |
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Institution: | .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 ;.Genome Informatics Laboratory, Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, 1111 Yata, Mishima, 411-8540 Shizuoka Japan |
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Abstract: | BackgroundHuman 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.ResultsHere 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-645) contains supplementary material, which is available to authorized users. |
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Keywords: | HLA Next-generation sequencing |
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