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的多位点基因（例如存在于主要组织相容性复合体（major histocompatibility complex, MHC）中的基因）进行特征描述与群体遗传分析，向来颇具挑战。传统的大规模克隆与桑格测序（Sanger sequencing）法不仅成本高昂且耗时费力，而间接评估方法往往会低估总变异水平。本研究探讨了454焦磷酸测序（454 pyrosequencing）用于群体遗传研究中多位点基因特征描述的适用性。我们以狼獾（Gulo gulo）的MHC DRB基因外显子2的185 bp片段为研究对象进行特征分析，对比了两种样本标记方案与两种用于454测序的生物信息学分析流程，并将所得结果与克隆及桑格测序的结果进行了比较。在检测的88个个体中，我们共发现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%的序列变异通常为测序假象。但我们发现，除多位点系统本身的复杂性外，样本制备与数据处理流程也会对变异频率产生显著影响。因此，我们建议针对每个独特的研究体系，确定专属的扩增子特异性变异频率阈值。本研究获得了极高的测序覆盖度（约5000×），结合焦磷酸测序的半定量特性，我们得以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实现这一点。我们所建立的基因座特异性MHC基因型获取方法，将有助于提升非模式野生动物物种的群体遗传分析与疾病易感性相关研究的质量。