title: Impact of three Illumina library construction methods on GC bias and HLA genotype calling.
abstract: 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.
authors.affiliation: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, USA; University of British Columbia, Clinician Investigator Program, Vancouver, BC, Canada.
authors.forename: James H
authors.initials: JH
authors.lastname: Lan
authors.affiliation: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, USA.
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Yin
authors.affiliation: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, USA.
authors.forename: Elaine F
authors.initials: EF
authors.lastname: Reed
authors.affiliation: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Moua
authors.affiliation: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, USA.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Thomas
authors.affiliation: UCLA Immunogenetics Center, Department of Pathology & Laboratory Medicine, Los Angeles, CA, USA.
authors.forename: Qiuheng
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D000483:Alleles; D003362:Cost-Benefit Analysis; D015723:Gene Library; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006650:Histocompatibility Testing / Q000191:economics / Q000379:methods; D006801:Humans; D015203:Reproducibility of Results; D019895:Transposases / Q000378:metabolism
chemical_list: D006680:HLA Antigens; D019895:Transposases
keywords: GC bias; HLA genotyping; Illumina Nextera; NGS; Transposase