Journal
HUMAN IMMUNOLOGY
Volume 76, Issue 2-3, Pages 166-175Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.humimm.2014.12.016
Keywords
HLA genotyping; NGS; Transposase; Illumina Nextera; GC bias
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Funding
- University of British Columbia Clinician Investigator Program
- NIH/National Center for Advancing Translational Science (NCATS) UCLA CTSI Grant [UL1TR000124]
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Next-generation sequencing (NGS) is increasingly recognized for its ability to overcome allele ambiguity and deliver high-resolution typing in the HLA system. Using this technology, non-uniform read distribution can impede the reliability of variant detection, which renders high-confidence genotype calling particularly difficult to achieve in the polymorphic HLA complex. Recently, library construction has been implicated as the dominant factor in instigating coverage bias. To study the impact of this phenomenon on HLA genotyping, we performed long-range PCR on 12 samples to amplify HLA-A, -B, -C, -DRB1, and -DQB1, and compared the relative contribution of three Illumina library construction methods (TruSeq Nano, Nextera, Nextera XT) in generating downstream bias. Here, we show high GC% to be a good predictor of low sequencing depth. Compared to standard TruSeq Nano, GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles. Importantly, our findings demonstrate non-uniform read depth can have a direct and negative impact on the robustness of HLA genotyping, which has clinical implications for users when choosing a library construction strategy that aims to balance cost and throughput with data quality. (C) 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
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