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VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma
Published on: December 28, 2015
Phase-defined complete sequencing of the HLA genes by next-generation sequencing
Kazuyoshi Hosomichi1, Timothy A Jinam, Shigeki Mitsunaga
1Division of Human Genetics, National Institute of Genetics, Shizuoka, Japan.
BMC Genomics
|May 30, 2013
Summary
A new method provides accurate, cost-effective, phase-defined sequencing for all human leukocyte antigen (HLA) genes. This advances high-resolution HLA typing and novel allele discovery for over 100 associated diseases.
Area of Science:
- Genomics
- Immunogenetics
Background:
- The human leukocyte antigen (HLA) region is crucial in immune response and associated with over 100 diseases, predominantly autoimmune conditions.
- Conventional sequencing methods face challenges in fully resolving complex HLA gene sequences and haplotype structures.
Purpose of the Study:
- To develop a novel, accurate, and cost-effective method for phase-defined complete sequencing of HLA genes.
- To enable high-resolution HLA typing and identification of rare or novel alleles.
Main Methods:
- Employed long-range PCR to amplify six key HLA genes (HLA-A, -C, -B, DRB1, -DQB1, -DPB1).
- Utilized transposase-based library construction and indexed multiplex next-generation sequencing (NGS) on a MiSeq platform.
- Developed distinct tagging strategies for homozygous and heterozygous samples to generate phase-defined sequences from promoter to 3'-UTR.
Main Results:
- Successfully generated phase-defined sequences for all six targeted HLA genes across homozygous, heterozygous, and parent-child trio samples.
- Achieved high-resolution HLA typing, enabling the assignment of up to 8-digit HLA allele numbers.
- Validated the accuracy and phasing capability of the method through parent-child trio sequencing.
Conclusions:
- The developed protocol provides comprehensive, phase-defined sequencing of entire HLA genes.
- This method significantly enhances HLA typing resolution and facilitates the detection of novel HLA alleles.
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