Mitochondrial DNA enrichment reduced NUMT contamination in porcine NGS analyses
Mitochondrial DNA enrichment reduced NUMT contamination in porcine NGS analyses
复制标题
线粒体 DNA 富集减少了猪 NGS 分析中的 NUMT 污染。
DOI：
10.1093/bib/bbz060
复制
发表时间：
2020-07-01
影响因子：
9.5
通讯作者：
Zhao，Xingbo
中科院分区：
文献类型：
--
作者：
Wang，Dan;Xiang，Hai;Zhao，Xingbo
线粒体DNA （mtDNA）与猪经济性状之间的遗传关联已被广泛报道，这表明mtDNA的重要性。然而，关于猪mtDNA异质性的研究很少。下一代测序（NGS）方法已成为检测线粒体异质性的一种有前途的基因组方法。由于短读数，灵活的生物信息学分析和核线粒体序列（NUMTs）的污染，NGS预计会增加异质性的假阳性检测。本研究采用Sanger测序作为金标准检测猪的异质，检测灵敏度为5%，然后采用一种全基因组测序方法（WGS）和两种mtDNA富集测序方法（Capture和LongPCR）。本研究的目的是确定NGS数据的线粒体异质性鉴定是否受到numt的影响。我们发现，与两种富集测序方法相比，WGS产生了更多的假个体内多态性和更低的定位特异性，这表明numt确实导致了NGS数据中线粒体异质的假阳性。此外，为了准确检测线粒体多样性，我们比较了samtools、VarScan和gatk三种常用的工具，它们具有不同的参数值。在考虑碱基比对质量重新计算和最小变异频率为0.25时，VarScan具有最佳的特异性和灵敏度。这也表明生物信息学工作流程干扰了mtDNA snp的鉴定。综上所述，来自NGS数据的猪线粒体的个体内多态性与numt相混淆，在检测线粒体基因组序列之前，mtdna特异性富集是必不可少的。
Genetic associations between mitochondrial DNA (mtDNA) and economic traits have been widely reported for pigs, which indicate the importance of mtDNA. However, studies on mtDNA heteroplasmy in pigs are rare. Next generation sequencing (NGS) methodologies have emerged as a promising genomic approach for detection of mitochondrial heteroplasmy. Due to the short reads, flexible bioinformatic analyses and the contamination of nuclear mitochondrial sequences (NUMTs), NGS was expected to increase false-positive detection of heteroplasmy. In this study, Sanger sequencing was performed as a gold standard to detect heteroplasmy with a detection sensitivity of 5% in pigs and then one whole-genome sequencing method (WGS) and two mtDNA enrichment sequencing methods (Capture and LongPCR) were carried out. The aim of this study was to determine whether mitochondrial heteroplasmy identification from NGS data was affected by NUMTs. We find that WGS generated more false intra-individual polymorphisms and less mapping specificity than the two enrichment sequencing methods, suggesting NUMTs indeed led to false-positive mitochondrial heteroplasmies from NGS data. In addition, to accurately detect mitochondrial diversity, three commonly used tools—SAMtools, VarScan and GATK—with different parameter values were compared. VarScan achieved the best specificity and sensitivity when considering the base alignment quality re-computation and the minimum variant frequency of 0.25. It also suggested bioinformatic workflow interfere in the identification of mtDNA SNPs. In conclusion, intra-individual polymorphism in pig mitochondria from NGS data was confused with NUMTs, and mtDNA-specific enrichment is essential before high-throughput sequencing in the detection of mitochondrial genome sequences.