Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage

Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time-intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185-bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per-amplicon-variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi-quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus-specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species.针对非模式脊椎动物，对多基因座基因（如主要组织相容性复合体（MHC）中的相关基因）开展特征描述与种群遗传学分析，历来颇具挑战。传统的大规模克隆与桑格测序（Sanger sequencing）方法不仅成本高昂、耗时费力，间接评估手段还往往会低估总变异水平。本研究探讨了454焦磷酸测序（454 pyrosequencing）用于种群遗传学研究中的多基因座基因特征描述的适用性。我们通过对貂熊（Gulo gulo）MHC DRB基因外显子2一段185 bp的片段进行特征分析，比较了两种样本标记方案与两种生物信息学流程在454测序中的应用效果，并将所得结果与克隆及桑格测序的结果进行了对比。我们在88个受检个体中发现了10个推定DRB等位基因，每个个体携带2至4个等位基因，这提示存在一个发生了复制的DRB基因。除推定等位基因外，所有个体均携带一个易于识别的假基因。在本研究体系中，单个样本中频率低于6%的序列变异通常为人工伪影。不过我们发现，除多基因座系统本身的复杂性外，样本制备与数据处理流程也会极大影响变异频率。因此，我们建议针对每个独特的研究体系设定每扩增子-变异体频率阈值。本研究获得的极高测序覆盖度（约5000×）结合焦磷酸测序的半定量特性，使我们能够将所有推定等位基因分配至两个DRB基因座，而传统方法通常无法做到这一点。我们这套获取座位特异性MHC基因型的方法，将有效助力非模式野生生物物种的种群遗传学分析与疾病易感性相关研究。