This document reports on a research study aimed at evaluating the newly developed massively parallel sequencing instrumentation, Thermo Fisher’s Precision ID GlobalFiler™ NGS STR Panel on their Ion S5 system, for forensic casework and DNA analysis.
This report, produced by the City of New York, discusses Thermo Fisher’s Precision ID GlobalFiler™ NGS STR Panel on their Ion S5 system for forensic casework, as part of an effort by the forensic community to evaluate the newly developed instrumentation that utilizes massively parallel sequencing (MPS), with the hopes of addressing the limitations of current technologies and improving throughput at lower costs. The GlobalFiler™ system testing pursued five specific goals: to determine accuracy and sensitivity using single source samples; to assess reliability, barcoding, and contamination; to test challenging samples, including degraded samples from bones, blood cards, and teeth, as well as mixed samples in various ratios from two to six people; to establish a high throughput liquid handling system for consistent library preparations; and to calculate costs and time from preparation through analysis. The outcomes of this testing were: verification of concordance for all DNA samples tested to the overlapping loci of PowerPlex Fusion and ForenSeq DNA signature Prep kit. Some samples with 50pg input revealed full profiles, repeatedly. Reducing the sample number per chip increased read numbers, while on samples with limited DNA, input decreased the number of drop-outs. Sample-specific barcodes prevented cross-contamination between samples while they were pooled for the sequencing reaction. Recovery PCRs performed on libraries had a positive effect on DNA samples with limited DNA input. The GlobalFiler™ NGS STR Panel v2 was not much affected by degraded DNA. All expected alleles were obtained with significant degraded DNA samples. Data from two-person mixtures showed drop-outs of the minor contributor; the higher the ratio, the more drop-outs were detected. Mixed samples with a ratio of 1:20 could barely be identified as mixed. The total GlobalFiler™ NGS STR Panel v2 is about 60h and the costs for one plate (32 samples) were calculated as being $5,029.
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