Abstract
Next-generation sequencing (NGS) of HLA class I and II loci (HLA-A, HLA-B, HLA-C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1) is described here in detail using the 454 Life Sciences GS FLX System and Titanium chemistry. An overview of the protocol with our experience on sequence performance efficiencies, read depth and ambiguity analyses using the GS FLX System are also presented. A total of 14 HLA primer pairs with multiplex identifiers (MIDs) are used in clonal, amplicon-based pyrosequencing of up to 44 samples per plate using the GS FLX. Genotype assignment and ambiguity reduction analysis is performed using Conexio Assign ATF 454 software. Clonal NGS gives a significant reduction in genotyping ambiguity during analysis of the highly complex HLA system.
Elizabeth A. Trachtenberg and Cherie L. Holcomb have contributed equally to this manuscript.
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Acknowledgments
The authors would like to thank Henry Erlich, Martha Ladner, David Noonan, and Melinda Rastrou for their thoughtful reading of the manuscript. We would also like to acknowledge Damian Goodridge for the development of the genotyping software, and Kazu Osoegawa, Martha Ladner, Franziska Cohen, Sherry Hawbecker, David Noonan for setting this system up and sequencing all the samples in the CHORI lab. We would also like to acknowledge Priscilla Moonsamy and Bryan Hoglund for their contributions to initial development of primers and protocols. E. Trachtenberg is supported in part by NIH grants 2UO1AI067068 and 2P01CA111412.
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Trachtenberg, E.A., Holcomb, C.L. (2013). Next-Generation HLA Sequencing Using the 454 GS FLX System. In: Zachary, A., Leffell, M. (eds) Transplantation Immunology. Methods in Molecular Biology, vol 1034. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-493-7_10
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DOI: https://doi.org/10.1007/978-1-62703-493-7_10
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