McNulty Samantha N, Mann Patrick R, Robinson Joshua A, Duncavage Eric J, Pfeifer John D
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri.
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri.
J Mol Diagn. 2020 May;22(5):720-727. doi: 10.1016/j.jmoldx.2020.02.003. Epub 2020 Mar 4.
PCR amplification, a key step in next-generation sequencing (NGS) library construction, can generate an unlimited amount of product from limited input; however, it cannot create more information than was present in the original template. Thus, NGS libraries can be made from very little DNA, but reducing the input may compromise assay sensitivity in ways that are difficult to ascertain unless library complexity (ie, the number of unique DNA molecules represented in the library) and depth of coverage with unique sequence reads (those derived from input DNA molecules) versus duplicate sequence reads (those resulting from overamplification of particular molecules) are discretely measured. A series of experiments was performed to explore the impact of low DNA input on an amplicon-based NGS assay using unique molecular identifiers to track unique versus duplicate reads. At high sequencing depths, unique and total (unique plus duplicate) read coverage are not well correlated, so increasing the number of sequenced reads does not necessarily improve sensitivity. Unique coverage depth tends to improve with more input, but improvements are not consistent. Fluctuations in library complexity complicated variant detection using both standardized and clinical specimens, often resulting in technical replicates with vastly different estimates of variant allelic fraction. In conclusion, depth of coverage with unique reads must be tracked in clinical NGS to ensure that sensitivity and accuracy are maintained.
聚合酶链式反应（PCR）扩增是二代测序（NGS）文库构建中的关键步骤，它能够从有限的起始材料中产生无限量的产物；然而，它无法创造出比原始模板中更多的信息。因此，可以用极少的DNA构建NGS文库，但减少起始材料的量可能会以难以确定的方式影响检测灵敏度，除非分别测定文库复杂度（即文库中所代表的独特DNA分子的数量）以及独特序列读数（源自起始DNA分子）与重复序列读数（特定分子过度扩增产生的读数）的覆盖深度。我们进行了一系列实验，以探讨低起始DNA量对基于扩增子的NGS检测的影响，该检测使用独特分子标识符来追踪独特读数与重复读数。在高测序深度下，独特读数覆盖度与总（独特读数加重复读数）覆盖度的相关性不佳，因此增加测序读数的数量不一定能提高检测灵敏度。独特覆盖深度往往会随着起始材料量的增加而改善，但改善情况并不一致。文库复杂度的波动使得使用标准化样本和临床样本进行变异检测变得复杂，常常导致技术重复之间的变异等位基因频率估计值差异很大。总之，在临床NGS中必须追踪独特读数的覆盖深度，以确保维持检测的灵敏度和准确性。
