检索高质量的内源性古DNA(aDNA)提出了几个挑战,包括低分子拷贝数,高碎片率,读取终端处的损坏,和潜在的外源污染物DNA的存在。所有这些因素使来自高通量测序平台的读取中的共有aDNA序列的可靠重建复杂化。这里,我们报告了对旨在克服这些问题并构建高质量古代有丝分裂基因组的两种替代工具(ANGSD和schmutzi)进行全面评估的结果.原始基因组数据(BAM/FASTQ)来自17个先前发表的整个古代人类基因组,范围从公元前14年到7年,并使用不同质量的过滤器重建线粒体共有序列。与他们的精度测量和比较。此外,不同序列参数的影响(读取次数,测序碱基,平均覆盖率,以及脱氨和污染率)作为衍生序列质量的预测因子进行了评估。所有古代样本都成功重建了完整的有丝分裂基因组,对于他们中的大多数人来说,过滤大大提高了mtDNA共识调用和单倍群预测。总的来说,schmutzi管道,它估计并考虑到外源污染,在中等至高覆盖率样品(>1,000,000个读数)中,似乎具有快得多且用户友好的替代方法(ANGSD)的优势。ANGSD,然而,通过它的读取终端微调过滤器,在从低质量样品中调用共有序列方面表现出更好的能力。在检查的所有总体样本质量的预测因子中,对于序列读数和碱基的可用数量,发现最强的相关性.在这个过程中,我们报告了来自南安纳托利亚/北黎凡特的早期石器病个体的先前未分配的单倍群(U3b)。
Retrieving high-quality endogenous ancient DNA (aDNA) poses several challenges, including low molecular copy number, high rates of fragmentation, damage at read termini, and potential presence of exogenous contaminant DNA. All these factors complicate a reliable reconstruction of
consensus aDNA sequences in reads from high-throughput sequencing platforms. Here, we report findings from a thorough evaluation of two alternative tools (ANGSD and schmutzi) aimed at overcoming these issues and constructing high-quality ancient mitogenomes. Raw genomic data (BAM/FASTQ) from a total of 17 previously published whole ancient human genomes ranging from the 14th to the 7th millennium BCE were retrieved and mitochondrial
consensus sequences were reconstructed using different quality filters, with their accuracy measured and compared. Moreover, the influence of different sequence parameters (number of reads, sequenced bases, mean coverage, and rate of deamination and contamination) as predictors of derived sequence quality was evaluated. Complete mitogenomes were successfully reconstructed for all ancient samples, and for the majority of them, filtering substantially improved
mtDNA consensus calling and haplogroup prediction. Overall, the schmutzi pipeline, which estimates and takes into consideration exogenous contamination, appeared to have the edge over the much faster and user-friendly alternative method (ANGSD) in moderate to high coverage samples (>1,000,000 reads). ANGSD, however, through its read termini trimming filter, showed better capabilities in calling the
consensus sequence from low-quality samples. Among all the predictors of overall sample quality examined, the strongest correlation was found for the available number of sequence reads and bases. In the process, we report a previously unassigned haplogroup (U3b) for an Early Chalcolithic individual from Southern Anatolia/Northern Levant.