Impact of three Illumina library construction methods on GC bias and HLA genotype calling.
Impact of three Illumina library construction methods on GC bias and HLA genotype calling.
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DOI：
10.1016/j.humimm.2014.12.016
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发表时间：
2015-03
期刊：
影响因子：
2.7
通讯作者：
Zhang, Qiuheng
中科院分区：
文献类型：
--
作者：
Lan, James H.;Yin, Yuxin;Reed, Elaine F.;Moua, Kevin;Thomas, Kimberly;Zhang, Qiuheng
下一代测序(NGS)因其克服等位基因歧义和在人类白细胞抗原(HL A)系统中提供高分辨率分型的能力而越来越受到人们的认可。使用这项技术时，不均匀的读数分布会阻碍变异检测的可靠性，这使得在多态的HLA复合体中实现高置信度的基因呼叫尤其困难。最近，图书馆建设被认为是引发报道偏差的主要因素。为了研究这一现象对人类白细胞抗原基因分型的影响，我们对12个样本进行了长程聚合酶链式反应，扩增了人类白细胞抗原-A、-B、-C、-DRB1和-DQB1，并比较了三种Illumina文库构建方法(TruSeq Nano、NextEra、NextEra XT)在产生下游偏差方面的相对贡献。在这里，我们表明高GC%是一个很好的预测低测序深度的指标。与标准的TruSeq Nano相比，GC偏倚在基于转座酶的方案中更加突出，特别是NextEra XT，可能是通过转座酶插入偏向与大量的PCR浓缩循环相结合的方式。重要的是，我们的研究结果表明，不均匀的读取深度会对人类白细胞抗原基因分型的稳健性产生直接和负面的影响，这对用户在选择旨在平衡成本和吞吐量与数据质量的文库构建策略时具有临床意义。
Next-generation sequencing (NGS) is increasingly recognized for its ability to overcome allele ambiguity and deliver high-resolution typing in the human leukocyte antigen (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.
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