de la Puente M, Phillips C, Santos C, Fondevila M, Carracedo Á, Lareu M V
Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain.
Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain.
Forensic Sci Int Genet. 2017 May;28:35-43. doi: 10.1016/j.fsigen.2017.01.012. Epub 2017 Jan 27.
A new forensic 140-SNP genotyping system from Qiagen, designed for massively parallel sequencing (MPS) analysis, was evaluated using the Ion PGM™ MPS system. Assessments consisted of the sequencing of: established control DNAs that had been previously genotyped with alternative PCR and library preparation kits supplied by Thermo Fisher Scientific for the Ion PGM™ system; a simple set of artificial DNA mixtures; DNA extracted from a degraded femur; and a dilution series to gauge forensic sensitivity. In addition to the reagents for the DNA target capture PCR and library preparation, Qiagen offer an alternative sequence analysis software system (Workbench), which was assessed alongside the Ion PGM™ Genotyper software for forensic MPS analysis. The Qiagen SNP genotyping system produced full genotyping concordance with previous data obtained with a similar SNP panel on the Ion PGM™ and in comparison to genotypes listed for 139 of the 140 SNPs in 1000 Genomes. The workbench software was as reliable as Genotyper in calling genotypes, although scrutiny of sequence data with IGV revealed the problem of sequence misalignment plagues a small proportion of the 140 SNPs in the Qiagen panel, a problem already recognized in multiple MPS studies of the same markers in alternative kits. The potential for genotype miscalls from sequence misalignment in certain SNPs will require manual inspection in cases where low-level or degraded DNA reduces the sequence coverage to a point where misalignment influences individual SNP genotype quality.
使用Ion PGM™ MPS系统对Qiagen公司一款全新的用于大规模平行测序（MPS）分析的法医140-SNP基因分型系统进行了评估。评估内容包括对以下样本进行测序：已使用赛默飞世尔科技公司为Ion PGM™系统提供的替代PCR和文库制备试剂盒进行过基因分型的既定对照DNA；一组简单的人工DNA混合物；从一根降解的股骨中提取的DNA；以及一个用于评估法医灵敏度的稀释系列。除了DNA靶标捕获PCR和文库制备试剂外，Qiagen公司还提供了一个替代序列分析软件系统（Workbench），并与用于法医MPS分析的Ion PGM™ Genotyper软件一起进行了评估。Qiagen SNP基因分型系统与之前在Ion PGM™上使用类似SNP面板获得的数据以及与1000基因组中140个SNP中的139个所列出的基因型完全一致。Workbench软件在基因型判读方面与Genotyper一样可靠，尽管使用IGV对序列数据进行仔细检查发现，Qiagen面板中的140个SNP中有一小部分存在序列比对错误的问题，这一问题在使用替代试剂盒对相同标记进行的多项MPS研究中已经被发现。在某些SNP中，由于序列比对错误导致基因型误判的可能性，在低水平或降解DNA将序列覆盖度降低到比对错误影响单个SNP基因型质量的程度时，将需要进行人工检查。
