Improved resolution of massively parallel sequencing (MPS) allows for the characterization of mitochondrial (mt) DNA heteroplasmy to levels previously unattainable with traditional sequencing approaches. An essential criterion for the reporting of heteroplasmy is the ability of the MPS method to distinguish minor sequence variants (MSVs) from system noise, or error. Therefore, an assessment of the background noise in the MPS method is desirable to identify the point at which reliable data can be reported. Substitution and sequence specific error (SSE) was evaluated for a variety of sample types and two library preparations. Substitution error rates ranged from 0.18-0.49 per 100 nucleotides with C positions generally having the highest rate of misincorporation. Comparison of error rates across sample types indicated a significant increase for samples with damaged DNA. The positions of error were varied across datasets (pairwise concordance 0-68%), but had greater consistency within the damaged samples (80-96%). The most commonly observed motif preceding error in forward reads was CCG, while GGT was most common in reverse reads, both consistent with previous findings. The findings illustrate that for datasets containing samples with damaged DNA, reporting thresholds for heteroplasmy may have to be modified and individual sites with error levels exceeding thresholds should be scrutinized. Collectively, the shifting error profiles observed across the various sample types and library preparation methods demonstrates the need for an assessment of error under these varying circumstances. Characterization of the applicable background noise will help to ensure that thresholds are reliably set for detection of true MSVs.

中文翻译：

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对来自各种样品来源和文库制备方法的人类线粒体 DNA 进行测序时 MiSeq MPS 数据中背景噪声的表征

大规模并行测序 (MPS) 分辨率的提高允许将线粒体 (mt) DNA 异质性表征到以前传统测序方法无法达到的水平。报告异质性的一个基本标准是 MPS 方法从系统噪声或错误中区分次要序列变异 (MSV) 的能力。因此，需要对 MPS 方法中的背景噪声进行评估，以确定可以报告可靠数据的点。评估了多种样品类型和两种文库制备的替代和序列特异性错误 (SSE)。替换错误率范围为每 100 个核苷酸 0.18-0.49，其中 C 位置通常具有最高的错误掺入率。不同样本类型的错误率比较表明，DNA 受损样本的错误率显着增加。错误的位置在数据集之间有所不同（成对一致性 0-68%），但在损坏的样本中具有更大的一致性（80-96%）。在正向读取中最常观察到的模体前置错误是 CCG，而 GGT 在反向读取中最常见，两者都与之前的发现一致。研究结果表明，对于包含 DNA 受损样本的数据集，可能必须修改异质性的报告阈值，并且应该仔细检查错误水平超过阈值的单个站点。总的来说，在各种样本类型和文库制备方法中观察到的移动错误概况表明需要在这些不同的情况下评估错误。