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Hum Immunol. 2015 Dec;76(12):917-22. doi: 10.1016/j.humimm.2015.04.007. Epub 2015 May 28.

Development and validation of a sample sparing strategy for HLA typing utilizing next generation sequencing.

Author information

1
Department of Vaccine Development, La Jolla Institute for Allergy and Immunology, 9820 Athena Circle, La Jolla, CA 92037, USA.
2
Bioinformatics Core Facility, La Jolla Institute for Allergy and Immunology, 9820 Athena Circle, La Jolla, CA 92037, USA.
3
Department of Vaccine Development, La Jolla Institute for Allergy and Immunology, 9820 Athena Circle, La Jolla, CA 92037, USA. Electronic address: alex@liai.org.

Abstract

We report the development of a general methodology to genotype HLA class I and class II loci. A Whole Genome Amplification (WGA) step was used as a sample sparing methodology. HLA typing data could be obtained with as few as 300 cells, underlining the usefulness of the methodology for studies for which limited cells are available. The next generation sequencing platform was validated using a panel of cell lines from the International Histocompatibility Working Group (IHWG) for HLA-A, -B, and -C. Concordance with the known, previously determined HLA types was 99%. We next developed a panel of primers to allow HLA typing of alpha and beta chains of the HLA DQ and DP loci and the beta chain of the DRB1 locus. For the beta chain genes, we employed a novel strategy using primers in the intron regions surrounding exon 2, and the introns surrounding exons 3 through 4 (DRB1) or 5 (DQB1 and DPB1). Concordance with previously determined HLA Class II types was also 99%. To increase throughput and decrease cost, we developed strategies combining multiple loci from each donor. Multiplexing of 96 samples per run resulted in increases in throughput of approximately 8-fold. The pipeline developed for this analysis (HLATyphon) is available for download at https://github.com/LJI-Bioinformatics/HLATyphon.

KEYWORDS:

HLA typing; Next generation sequencing; Pipeline; Sample sparing

PMID:
26027778
PMCID:
PMC4662932
DOI:
10.1016/j.humimm.2015.04.007
[Indexed for MEDLINE]
Free PMC Article

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