Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird
Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird
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DOI：
10.1111/1755-0998.12612
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发表时间：
2017-07-01
影响因子：
7.7
通讯作者：
Radwan, Jacek
中科院分区：
文献类型：
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作者：
Biedrzycka, Aleksandra;Sebastian, Alvaro;Radwan, Jacek
高重复基因的表征，如主要组织相容性复合体（MHC）的基因，其中多个位点经常共同扩增，直到最近一直受到每个扩增子读取深度不足的阻碍。在这里，我们使用超深Illumina测序分析了沙莺（Acrocephalus schoenobaenus） MHC I类基因外显子3的基因型。我们在两个重复中对24个个体进行了测序，并使用该数据以及模拟数据集，测试了使用四种不同的基因分型方法，扩增子覆盖率（范围：每个扩增子500- 20,000 reads）对基因分型可重复性的影响。第三个重复使用独特的条形码来评估标签跳跃的程度，即个体标签标识符的交换，这可能会混淆基因分型。MHC基因分型的可靠性随着覆盖率的增加而增加，在每个扩增子覆盖5000个reads时，等位基因呼叫的方法内重复性接近或超过90%。我们发现基因分型方法之间普遍高度一致，特别是在高覆盖率的情况下。我们对模拟数据集的分析进一步支持了所测试的基因分型方法的高可靠性，尽管主要依赖于独立扩增子中变异的复制的基因分型方法被证明对可重复的错误很敏感。根据最可重复的基因分型方法，每个个体的共扩增变异数从19到42不等。标签跳跃是可以检测到的，但频率很低，不影响基因分型的可靠性。因此，我们证明具有许多共同扩增基因的基因家族可以使用HTS可靠地进行基因分型，前提是每个扩增子有足够的覆盖率。
Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of geno-typing using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage.