Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China.
Qitan Technology Ltd., Chengdu 610044, China.
Forensic Sci Int Genet. 2022 Mar;57:102657. doi: 10.1016/j.fsigen.2021.102657. Epub 2021 Dec 27.
In recent years, extraordinary progress has been made in genome sequencing technologies, which has led to a decrease in cost and an increase in the diversity of sequenced genomes. Nanopore sequencing is one of the latest genome sequencing technologies. It aims to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions, and provides a new approach for forensic genetics to detect longer markers in real time. To date, multiple studies have been conducted to sequence forensic markers using MinION from Oxford Nanopore Technologies (ONT), and the results indicate that nanopore sequencing holds promise for forensic applications. Qitan Technology (QitanTech) recently launched its first commercial nanopore genome sequencer, QNome. It could achieve a read length of more than 150 kbp, and could generate approximately 500 Mb of data in 8 h. In this pilot study, we explored and validated this alternative nanopore sequencing device for microhaplotype (MH) profiling using a custom set of 15 MH loci. Seventy single-contributor samples were divided into 7 batches, each of which included 10 samples and control DNA 9947A and was sequenced by QNome. MH genotypes generated from QNome were compared to those from Ion Torrent sequencing (Ion S5XL system) to evaluate the accuracy and stability. Twelve samples randomly selected from the last three batches and Control DNA 9947A were also subjected to ONT MinION sequencing (with R9.4 flow cell) for parallel comparison. Based on MHtyper, a bioinformatics workflow developed for automated MH designation, all MH loci can be genotyped and reliably phased using the QNome data, with an overall accuracy of 99.83% (4 errors among 2310 genotypes). Three occurred near or in the region of homopolymer sequences, and one existed within 50 bp of the start of the sequencing reaction. In the last 15 samples (12 individual samples and 3 replicates of control DNA 9947A), two SNPs located at 4-mer homopolymers failed to obtain reliable genotypes on the MinION data. This study shows the potential of state-of-the-art nanopore sequencing methods to analyze forensic MH markers. Given the rapid pace of change, sporadic and nonrepetitive errors presented in this study are expected to be resolved by further developments of nanopore technologies and analysis tools.
近年来，基因组测序技术取得了非凡的进展，测序成本降低，测序基因组的多样性增加。纳米孔测序是最新的基因组测序技术之一。它旨在对较长的连续 DNA 片段进行测序，这对于解决结构复杂的区域至关重要，并为法医遗传学提供了一种实时检测更长标记物的新方法。迄今为止，已经有多项研究使用牛津纳米孔技术公司（ONT）的 MinION 对法医标记物进行测序，结果表明纳米孔测序在法医应用中具有广阔的前景。奇点技术（QitanTech）最近推出了其首款商用纳米孔基因组测序仪 QNome。它可以实现超过 150 kbp 的读取长度，在 8 小时内可以生成大约 500 Mb 的数据。在这项初步研究中，我们使用一组 15 个微单倍型（MH）位点来探索和验证这种替代的纳米孔测序设备。70 个单份样本分为 7 批，每批包括 10 个样本和对照 DNA 9947A，并由 QNome 测序。从 QNome 生成的 MH 基因型与 Ion Torrent 测序（Ion S5XL 系统）生成的 MH 基因型进行比较，以评估准确性和稳定性。最后三批中随机选择的 12 个样本和对照 DNA 9947A 也被送到 ONT MinION 测序（使用 R9.4 流动池）进行并行比较。基于 MHtyper，这是一种用于自动 MH 命名的生物信息学工作流程，使用 QNome 数据可以对所有 MH 位点进行基因分型并可靠地定相，总体准确率为 99.83%（2310 个基因型中有 4 个错误）。三个错误发生在或靠近长串联重复序列区域，一个错误发生在测序反应开始的 50 个碱基内。在最后 15 个样本（12 个个体样本和 3 个对照 DNA 9947A 重复样本）中，两个位于 4 碱基长串联重复序列上的 SNP 无法在 MinION 数据上获得可靠的基因型。这项研究表明，最先进的纳米孔测序方法具有分析法医 MH 标记物的潜力。鉴于纳米孔技术和分析工具的快速发展，预计本研究中出现的偶发和非重复性错误将通过进一步的发展得到解决。
