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.
Highlights
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
All 96 PCR amplicons were subjected to transposase-based library construction using the Nextera kit, which simultaneously fragments the DNA and adds adaptors needed for multiplex sequencing
We developed protocol steps using altered AMPure XP beads to normalize the molar concentrations of 96 samples
Summary
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. We have previously developed a phase-defined HLA gene sequencing method using MiSeq sequencing. Several high-throughput HLA typing methods using next-generation sequencing (NGS) have been developed [1,2,3,4,5,6,7,8]. We completely sequenced long-range PCR amplicons encompassing entire regions of each of the HLA genes (HLA-A, −C, −B, −DRB1, −DQB1, and -DPB1). PCR amplicons were subjected to transposasebased library construction and multiplex sequencing with the MiSeq sequencer. Our sequencing protocol and pipeline provided essentially complete phase-defined HLA gene sequences; it required complicated and laborintensive workflows especially in the library preparation
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