利用高通量测序技术对多位点基因家族进行精确的基因分型,例如主要组织相容性复合体(MHC),仍然具有挑战性,由于数据的复杂性和区分真实和错误变体的困难。用于高通量测序数据的几个专用基因分型管道,例如下一代测序(NGS),已经被开发来解决随之而来的人为膨胀的多样性的风险。这里,我们彻底评估了NGS数据的三个这样的多基因座基因分型管道,DOC方法,AmpliSAS和ACACIA,使用MHCII类β数据集的三刺gDNA,具有已知等位基因多样性的cDNA和“人工”质粒样品。我们表明,在最佳管道参数下对gDNA和质粒样品进行基因分型是高度准确的,并且在各种方法中具有可重复性。然而,对于cDNA数据,gDNA最佳参数配置降低了总体基因分型精度和管道之间的一致性。需要进一步调整关键聚类参数,以说明每个等位基因的更高错误率和测序深度的更大变化。强调了模板特异性管道优化对多位点基因家族可靠基因分型的重要性。通过准确配对的gDNA-cDNA分型和MHC-II单倍型推断,我们显示MHC-II等位基因特异性表达水平与单倍型的等位基因数量呈负相关.最后,MHC-I的同胞辅助cDNA分型揭示了在单倍型区块中连锁的新变体,和高于以前报道的个体MHC-I等位基因多样性。总之,我们提供了新的基因分型方案三-棘刺MHC-I和-II基因,并评估流行的NGS基因分型管道的性能。我们还表明,配对gDNA-cDNA样品的微调基因分型有助于扩增偏倚校正的MHC等位基因表达分析。
Using high-throughput sequencing for precise genotyping of multi-locus gene families, such as the major histocompatibility complex (MHC), remains challenging, due to the complexity of the data and difficulties in distinguishing genuine from erroneous variants. Several dedicated genotyping pipelines for data from high-throughput sequencing, such as next-generation sequencing (NGS), have been developed to tackle the ensuing risk of artificially inflated diversity. Here, we thoroughly assess three such multi-locus genotyping pipelines for NGS data, the DOC method,
AmpliSAS and ACACIA, using MHC class IIβ data sets of three-spined stickleback gDNA, cDNA and \"artificial\" plasmid samples with known allelic diversity. We show that genotyping of gDNA and plasmid samples at optimal pipeline parameters was highly accurate and reproducible across methods. However, for cDNA data, the gDNA-optimal parameter configuration yielded decreased overall genotyping precision and consistency between pipelines. Further adjustments of key clustering parameters were required tο account for higher error rates and larger variation in sequencing depth per allele, highlighting the importance of template-specific pipeline optimization for reliable genotyping of multi-locus gene families. Through accurate paired gDNA-cDNA typing and MHC-II haplotype inference, we show that MHC-II allele-specific expression levels correlate negatively with allele number across haplotypes. Lastly, sibship-assisted cDNA-typing of MHC-I revealed novel variants linked in haplotype blocks, and a higher-than-previously-reported individual MHC-I allelic diversity. In conclusion, we provide novel genotyping protocols for the three-spined stickleback MHC-I and -II genes, and evaluate the performance of popular NGS-genotyping pipelines. We also show that fine-tuned genotyping of paired gDNA-cDNA samples facilitates amplification bias-corrected MHC allele expression analysis.