Sequencing of 231 forensic genetic markers using the MiSeq FGx™ forensic genomics system – an evaluation of the assay and software

The MiSeq FGx™ Forensic Genomics System types 231 genetic markers in one multiplex polymerase chain reaction (PCR) assay. The markers include core forensic short tandem repeats (STRs) as well as identity, ancestry and phenotype informative short nucleotide polymorphisms (SNPs). In this work, the MiSeq FGx™ Forensic Genomics System was evaluated by analysing reproducibility, sensitivity, mixture identification and forensic phenotyping capabilities of the assay. Furthermore, the genotype calling of the ForenSeq™ Universal Analysis Software was verified by analysing fastq.gz files from the MiSeq FGx™ platform using the softwares STRinNGS and GATK. Overall, the performance of the MiSeq FGx™ Forensic Genomics System was high. However, locus and allele drop-outs were relatively frequent at six loci (two STRs and four human identification SNPs) due to low read depth or skewed heterozygote balances, and the stutter ratios were larger than those observed with conventional STR genotyping methods. The risk of locus and allele drop-outs increased dramatically when the amount of DNA in the first PCR was lower than 250 pg. Two-person 50:1 mixtures were identified as mixtures, whereas 100:1 and 1 000:1 mixtures were not. Y-chromosomal short tandem repeats (Y-STRs) alleles were detected in the 100:1 and 1 000:1 female/male mixtures. The ForenSeq™ Universal Analysis Software provided the data analyst with useful alerts that simplified the analysis of the large number of markers. Many of the alerts were due to user-defined, locus-specific criteria. The results shown here indicated that the default settings should be altered for some loci. Also, recommended changes to the assay and software are discussed.

MiSeq FGx™ 法医基因组学系统可通过单次多重聚合酶链式反应（PCR）检测体系，对231个遗传标记完成分型。该系统涵盖的标记包括核心法医短串联重复序列（STRs），以及用于身份识别、祖源分析与表型预测的信息性单核苷酸多态性（SNPs）。本研究通过分析该检测体系的重复性、灵敏度、混合样本识别能力与法医表型分型性能，对MiSeq FGx™ 法医基因组学系统进行了全面评估。此外，本研究借助STRinNGS与GATK软件，对MiSeq FGx™ 平台产出的fastq.gz文件进行分析，验证了ForenSeq™ 通用分析软件（ForenSeq™ Universal Analysis Software）的基因型检出准确性。整体而言，MiSeq FGx™ 法医基因组学系统的分型性能优异。但由于测序读深较低或杂合子平衡偏移，6个遗传位点（2个STRs与4个人类身份识别SNPs）出现位点与等位基因丢失的频率相对较高，且其滑动峰比例高于传统STR分型方法的检测结果。当首轮PCR的模板DNA投入量低于250 pg时，位点与等位基因丢失的风险会显著升高。50:1比例的二人混合样本可被准确识别为混合样本，但100:1与1000:1比例的混合样本则无法被正确识别。在100:1与1000:1比例的女/男混合样本中，均检出了Y染色体短串联重复序列（Y-STRs）的等位基因。ForenSeq™ 通用分析软件可向数据分析人员提供实用的告警提示，简化了海量遗传标记的分析流程，其中多数告警基于用户自定义的位点特异性判定标准生成。本研究结果显示，部分位点的默认分析参数需要进行调整。此外，本文还讨论了针对该检测体系与分析软件的优化改进建议。