Department of Forensic Sciences, The George Washington University, Washington, DC, USA.
Methods Mol Biol. 2023;2685:397-427. doi: 10.1007/978-1-0716-3295-6_24.
Sequencing forensic DNA samples that are amplified and prepared with the ForenSeq™ DNA Signature Prep Kit allows for the simultaneous targeting of forensically relevant STR and SNP markers. The MiSeq™ FGx system allows massively parallel sequencing of these markers in a single analysis. The library preparation targets autosomal, Y-, and X-STRs, as well as identity SNPs. The kit can also be used to generate investigative information regarding the DNA contributor by analyzing phenotypic SNPs to predict hair color, eye color, and ancestry SNPs.Through two rounds of amplification, all loci are amplified and tagged with individualizing barcodes for sequencing capture and identification. Using bead-based technology, the libraries are purified by the removal of left-over amplification reagents and then normalized to ensure equal representation of all samples during sequencing. The individual libraries are then pooled for insertion into the MiSeq FGx. The pooled libraries are then added to a pre-packaged cartridge that contains all reagents necessary for optimal sequencing. Libraries are captured on a flow cell and undergo bridge amplification for the generation of individual clusters. Sequencing of each cluster is performed using a Sequence-By-Synthesis technology. The following chapter describes the methodology and process of library preparation of samples using the ForenSeq™ DNA Signature Prep Kit Primer Set A and B. Once completed, the chapter then focuses on the setup of a sequencing run on the MiSeq FGx and the sequencing methodology employed by the instrument.
对使用 ForenSeq™ DNA Signature Prep Kit 进行扩增和制备的法医 DNA 样本进行测序，可同时针对法医学相关 STR 和 SNP 标记物进行靶向分析。MiSeq™ FGx 系统允许在单次分析中对这些标记物进行大规模平行测序。文库制备针对常染色体、Y 染色体和 X 染色体 STR 以及身份 SNP。试剂盒还可以通过分析表型 SNP 来预测头发颜色、眼睛颜色和祖先 SNP，从而生成有关 DNA 供体的调查信息。通过两轮扩增，所有基因座都被扩增，并带有用于测序捕获和识别的个体化条形码。使用基于珠子的技术，通过去除多余的扩增试剂来纯化文库，然后对文库进行归一化处理，以确保在测序过程中所有样本的代表性均等。然后将各个文库混合，用于插入 MiSeq FGx。将混合文库添加到包含最佳测序所需的所有试剂的预制盒中。文库在流动槽上捕获，并进行桥接扩增，以生成单独的簇。使用测序合成技术对每个簇进行测序。以下章节描述了使用 ForenSeq™ DNA Signature Prep Kit Primer Set A 和 B 对样本进行文库制备的方法和流程。完成后，该章节将重点介绍在 MiSeq FGx 上设置测序运行以及该仪器所采用的测序方法。
