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¹Division of Human Genetics, National Institute of Genetics, 1111 Yata, Mishima, 411-8540 Shizuoka, Japan. itinoue@nig.ac.jp.

|August 5, 2014

Summary

We developed a cost-effective, high-throughput sequencing method for human leukocyte antigen (HLA) typing. This method enables accurate HLA-B allele phasing for 96 samples, crucial for disease association studies.

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Area of Science:

Genetics
Immunology
Molecular Biology

Background:

Human leukocyte antigen (HLA) genes are highly polymorphic and linked to over 100 diseases and drug effects.
Accurate HLA typing is vital for clinical applications, research, and population genetics.
Previous work established a phase-defined HLA gene sequencing method using MiSeq.

Purpose of the Study:

To report a simple, high-throughput, and cost-effective sequencing method for HLA typing.
To optimize HLA sequencing for multiplexing 96 samples.
To achieve reliable, high-resolution HLA-B allele phasing.

Main Methods:

Utilized long-range PCR to amplify HLA-B from 96 samples.
Employed transposase-based library construction and multiplex sequencing on a MiSeq platform.
Implemented normalized library preparation and DNA molar concentration adjustment.

Main Results:

Observed low variation (0.2%–1.55%) in read percentages across 96 demultiplexed samples.
Demonstrated successful haplotype phasing for all samples using the phase-defined sequencing method.
Determined that 800x sequencing depth is sufficient for full HLA-B phasing to the 8-digit level.

Conclusions:

The developed HLA sequencing method is time- and cost-effective for 96 multiplexed samples.
The method is suitable for automated multi-sample library preparation and sequencing.
This approach enhances the efficiency and reliability of HLA typing.