Data from: Large-scale genotyping of highly polymorphic loci by next generation sequencing: how to overcome the challenges to reliably genotype individuals?

Studying the different roles of adaptive genes is still a challenge in evolutionary ecology and requires reliable genotyping of large numbers of individuals. Next-generation sequencing (NGS) techniques enable such large-scale sequencing, but stringent data processing is required. Here, we develop an easy to use methodology to process amplicon-based NGS data and we apply this methodology to reliably genotype four major histocompatibility complex (MHC) loci belonging to MHC class I and II of Alpine marmots (Marmota marmota). Our post-processing methodology allowed us to increase the number of retained reads. The quality of genotype assignment was further assessed using three independent validation procedures. A total of 3069 high-quality MHC genotypes were obtained at four MHC loci for 863 Alpine marmots with a genotype assignment error rate estimated as 0.21%. The proposed methodology could be applied to any genetic system and any organism, except when extensive copy-number variation occurs (that is, genes with a variable number of copies in the genotype of an individual). Our results highlight the potential of amplicon-based NGS techniques combined with adequate post-processing to obtain the large-scale highly reliable genotypes needed to understand the evolution of highly polymorphic functional genes.

探究适应性基因的多样功能角色，仍是进化生态学领域的核心挑战之一，此类研究需对海量个体开展可靠的基因分型（genotyping）工作。下一代测序（next-generation sequencing，NGS）技术可为这类大规模测序工作提供支撑，但该流程需经过严格的数据处理环节。本研究开发了一款易用的分析方法，用于处理基于扩增子的NGS数据，并将该方法应用于对阿尔卑斯旱獭（Marmota marmota）的4类主要组织相容性复合体（major histocompatibility complex，MHC）基因座进行可靠分型，这些基因座隶属于MHC I类与MHC II类。我们所提出的后处理分析方法，有效提升了有效测序读段（reads）的保留数量。本研究进一步通过三项独立的验证流程，对基因分型结果的准确性进行了评估。本研究最终为863只阿尔卑斯旱獭的4个MHC基因座共获得3069条高质量MHC分型结果，经估算其基因分型错误率仅为0.21%。该方法可推广应用于任意遗传系统与生物物种，仅当研究对象存在广泛的拷贝数变异（copy-number variation）——即个体基因型内基因拷贝数存在可变数量的情况时除外。本研究结果证实，基于扩增子的NGS技术搭配合理的后处理流程，可获取大规模的高可靠基因分型数据，这为解析高度多态性功能基因的进化机制提供了有力支撑。