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Hum Immunol. 2015 Dec;76(12):897-902. doi: 10.1016/j.humimm.2015.08.002. Epub 2015 Aug 22.

Report on the effects of fragment size, indexing, and read length on HLA sequencing on the Illumina MiSeq.

Author information

1
ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132, United States. Electronic address: tracie.profaizer@aruplab.com.
2
ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132, United States.

Abstract

Single-molecule sequencing should allow for unambiguous, accurate, and high-throughput HLA typing. In this proof of principle study, we investigated the effects of fragment size for library preparation, indexing strategy, and read length on HLA typing. Whole gene amplicons of HLA-A, B, C, DRB1, and DQB1 were obtained by long-range PCR. For library preparation, two fragment sizes were evaluated: 100-300bp and 300-600bp. For sample multiplexing, two indexing strategies were compared: indexing-by-amplicon, where each individual amplicon is barcoded, and indexing-by-patient, where each patient's five loci are equimolarly pooled after PCR and indexed with the same barcode. Sequencing was performed on an Illumina MiSeq instrument using paired-end 150bp and 250bp read lengths. Our results revealed that the 300-600bp fragments in the 2×250 MiSeq group gave the most accurate sequencing results. There was no difference in HLA typing results between the two indexing strategies, suggesting that indexing-by-patient, which is much simpler, is a viable option. In conclusion, enzymatic fragmentation of pooled whole gene amplicons is a suitable strategy for HLA typing by next-generation sequencing on the Illumina MiSeq.

KEYWORDS:

HLA typing; Illumina MiSeq; Next-generation sequencing

PMID:
26303189
DOI:
10.1016/j.humimm.2015.08.002
[Indexed for MEDLINE]

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