Systematic assessment of the performance of Illumina's MiSeq FGx™ forensic genomics system

Abstract

This study assesses the performance of Illumina's MiSeq FGx System for forensic genomics by systematically analyzing single source samples, evaluating concordance, sensitivity and repeatability, as well as describing the quality of the reported outcomes. DNA from 16 individuals (9 males/7 females) in nine separate runs showed consistent STR profiles at DNA input ≥400 pg, and two full profiles were obtained with 50 pg DNA input. However, this study revealed that the outcome of a single sample does not merely depend on its DNA input but is also influenced by the total amount of DNA loaded onto the flow cell from all samples. Stutter and sequence or amplification errors can make the identification of true alleles difficult, particularly for heterozygous loci that show allele imbalance. Sequencing of 16 individuals' STRs revealed genetic variations at 14 loci at frequencies suggesting improvement of mixture deconvolution. The STR loci D1S1656 and DXS10103 were most susceptible to drop outs, and D22S1045 and DYS385a-b showed heterozygote imbalance. Most stutters were typed at TH01 and DYS385a-b, while amplification or sequencing errors were observed mostly at D7S820 and D19S433. Overall, Illumina's MiSeq FGx System produced reliable and repeatable results. aSTRs showed fewer drop outs than the Y- and X-STRs.

Inter-laboratory study on standardized MPS libraries: evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA

“…All markers showed HB ≥ 0.60 (manufacturer's default threshold settings) except for D22S1045 and D5S818 that yielded lower HB values, 0.43 (0.02) and 0.57 (0.02), respectively (Table S6). These findings were in line with earlier studies that reported imbalances at D22S1045 [3, 12-15, 17, 22, 54, 57] and D5S818 [53,54,57]. Six aSTRs (D4S2408, CSF1PO, D7S820, D8S1179, D9S1122 and TH01), one Y-STR (DYF387S1) and three X-STRs (DXS10135, DXS10074 and HPRTB) displayed HB ≥ 0.90 (Table S6).…”

Evaluation of ForenSeq™ Signature Prep Kit B on predicting eye and hair coloration as well as biogeographical ancestry by using Universal Analysis Software (UAS) and available web‐tools

“…Physical characteristics and biogeographical ancestry predictions can provide investigative leads in cases where no information in otherwise available . The ForenSeq™ DNA Signature Prep Kit using Primer Mix B was recently assessed for some performance metrics however, only one of these studies included phenotypic and ancestry predictions in their assessment of UAS (725 individuals tested) and found that ancestry and phenotypic predictions were obtainable for most samples.…”

“…Recently, the ForenSeq™ Signature Prep Kit has been evaluated for sensitivity, repeatability, allele coverage ratio, concordance with capillary electrophoresis methods, as well as aptitude in handling mixed DNA samples, challenging (degraded) samples, and ancient DNA samples . Additional tests for phenotypic and biogeographical ancestry predictions for 725 individuals from four population groups: Chinese, African American, US Caucasians, and Southwest Hispanics, found that ancestry and phenotype predictions were obtainable for most individual samples and phenotypic estimations were generally concordant with the population .…”

Qualitative and quantitative assessment of Illumina’s forensic STR and SNP kits on MiSeq FGx™

“…Recent evaluations of MPS kits and instruments for forensic DNA testing have shown that outcomes are, in general, concordant with CE methods and full profiles could be obtained from as little as 25 to 100 pg DNA. The reports included three HID-Ion AmpliSeq™ Identity Panels (Thermo Fisher Scientific) targeting over 100 identity SNPs (iSNPs) [ 11 – 13 ], two Ion PGM panels (Thermo Fisher Scientific) testing 10 autosomal STRs ( aSTRs ) [ 14 , 15 ], Promega’s panels testing 17 or 23 STR kits plus amelogenin [ 16 , 17 ], and Illumina’s MiSeq FGx platform testing their Primer Mix A (153 targets/loci, including 27 aSTRs, 24 Y-STRs, 7 X-STRs, and 94 iSNPs plus amelogenin), and/or Primer Mix B (231 loci, including Primer Mix A plus 22 phenotypic and 56 biogeographical ancestry SNPs) [ 18 – 23 ].…”