Applied Microbiology, Department of Chemistry, Lund University, SE-221 00, Lund, Sweden; Swedish National Forensic Centre, Swedish Police Authority, SE-581 94, Linköping, Sweden.
Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, 20899-8314, United States.
Forensic Sci Int Genet. 2019 May;40:182-191. doi: 10.1016/j.fsigen.2019.03.001. Epub 2019 Mar 2.
Massively parallel sequencing holds great promise for new possibilities in the field of forensic genetics, enabling simultaneous analysis of multiple markers as well as offering enhanced short tandem repeat allele resolution. A challenge in forensic DNA analysis is that the samples often contain low amounts of DNA in a background that may interfere with downstream analysis. PCR inhibition mechanisms of some relevant molecules have been studied applying e.g. real-time PCR and digital PCR. However, a detailed understanding of the effects of inhibitory molecules on forensic MPS, including mechanisms and ways to relieve inhibition, is missing. In this study, the effects of two well-characterized PCR inhibitors, humic acid and hematin, have been studied using the ForenSeq DNA Signature Prep kit. Humic acid and hematin resulted in lowered read numbers as well as specific negative effects on certain markers. Quality control of libraries with Fragment analyzer showed that increasing amounts of inhibitors caused a lowered amplicon quantity and that the larger amplicons were more likely to drop out. Further, the inhibitor tolerance could be improved 5-10 times by addition of bovine serum albumin in the initial PCR. On the contrary to the samples with inhibitors, low-template samples resulted in lowered read numbers for all markers. This difference strengthened the conclusion that the inhibitors have a negative effect on the DNA polymerase activity in the initial PCR. Additionally, a common capillary gel electrophoresis-based STR kit was shown to handle at least 200 times more inhibitors than the ForenSeq DNA Signature Prep kit. This suggests that there is room for improvement of the PCR components to ensure analytical success for challenging samples, which is needed for a broad application of MPS for forensic STR analysis.
高通量测序在法医遗传学领域具有巨大的应用潜力，能够同时分析多个标记物，并提供增强的短串联重复序列等位基因分辨率。法医 DNA 分析中的一个挑战是，样本中通常含有少量的 DNA，且背景中可能存在干扰下游分析的物质。已经应用实时 PCR 和数字 PCR 等方法研究了一些相关分子的 PCR 抑制机制。然而，对于法医高通量测序中抑制分子的影响，包括抑制机制和缓解抑制的方法，我们还缺乏详细的了解。在这项研究中，使用 ForenSeq DNA Signature Prep 试剂盒研究了两种特征明确的 PCR 抑制剂——腐殖酸和血红素的影响。腐殖酸和血红素导致读取数量降低，并对某些标记物产生特定的负面影响。片段分析仪的文库质量控制表明，随着抑制剂数量的增加，扩增子的数量降低，而且较大的扩增子更有可能丢失。此外，通过在初始 PCR 中添加牛血清白蛋白，可以将抑制剂的耐受能力提高 5-10 倍。与含有抑制剂的样本不同，低模板样本导致所有标记物的读取数量降低。这一差异进一步证实了抑制剂对初始 PCR 中 DNA 聚合酶活性有负面影响的结论。此外，基于毛细管凝胶电泳的 STR 试剂盒显示可以处理比 ForenSeq DNA Signature Prep 试剂盒多 200 倍的抑制剂。这表明有必要改进 PCR 组件，以确保具有挑战性的样本的分析成功，从而为法医 STR 分析中高通量测序的广泛应用提供支持。
