title: A Bead-based Normalization for Uniform Sequencing depth (BeNUS) protocol for multi-samples sequencing exemplified by HLA-B.
abstract: 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.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Hosomichi
authors.forename: Shigeki
authors.initials: S
authors.lastname: Mitsunaga
authors.forename: Hideki
authors.initials: H
authors.lastname: Nagasaki
authors.affiliation: Division of Human Genetics, National Institute of Genetics, 1111 Yata, Mishima, 411-8540 Shizuoka, Japan. itinoue@nig.ac.jp.
authors.forename: Ituro
authors.initials: I
authors.lastname: Inoue
mesh_terms: D003362:Cost-Benefit Analysis; D015235:HLA-B Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*
chemical_list: D015235:HLA-B Antigens
references.citation: Tissue Antigens. 2012 Oct;80(4):305-16
references.pmid: 22861646
references.citation: Tissue Antigens. 2009 Nov;74(5):393-403
references.pmid: 19845894
references.citation: Hum Immunol. 2010 Oct;71(10):1033-42
references.pmid: 20603174
references.citation: PLoS One. 2013 Nov 06;8(11):e78410
references.pmid: 24223151
references.citation: BMC Genomics. 2012 Aug 06;13:378
references.pmid: 22866951
references.citation: Genome Res. 2002 Apr;12(4):656-64
references.pmid: 11932250
references.citation: BMC Genomics. 2012 Jul 24;13:341
references.pmid: 22827831
references.citation: Brief Bioinform. 2013 Mar;14(2):178-92
references.pmid: 22517427
references.citation: DNA Res. 2013 Aug;20(4):383-90
references.pmid: 23657089
references.citation: BMC Genomics. 2013 May 28;14:355
references.pmid: 23714642
references.citation: Proc Natl Acad Sci U S A. 2012 May 29;109(22):8676-81
references.pmid: 22589303
references.citation: Hum Immunol. 2010 Oct;71(10):1011-7
references.pmid: 20650293
references.citation: BMC Genomics. 2011 Jan 18;12:42
references.pmid: 21244689