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

Abstract

Massively parallel sequencing (MPS) is a powerful tool transforming DNA analysis in multiple fields ranging from medicine, to environmental science, to evolutionary biology. In forensic applications, MPS offers the ability to significantly increase the discriminatory power of human identification as well as aid in mixture deconvolution. However, before the benefits of any new technology can be employed, a thorough evaluation of its quality, consistency, sensitivity, and specificity must be rigorously evaluated in order to gain a detailed understanding of the technique including sources of error, error rates, and other restrictions/limitations. This extensive study assessed the performance of Illumina’s MiSeq FGx MPS system and ForenSeq™ kit in nine experimental runs including 314 reaction samples. In-depth data analysis evaluated the consequences of different assay conditions on test results. Variables included: sample numbers per run, targets per run, DNA input per sample, and replications. Results are presented as heat maps revealing patterns for each locus. Data analysis focused on read numbers (allele coverage), drop-outs, drop-ins, and sequence analysis. The study revealed that loci with high read numbers performed better and resulted in fewer drop-outs and well balanced heterozygous alleles. Several loci were prone to drop-outs which led to falsely typed homozygotes and therefore to genotype errors. Sequence analysis of allele drop-in typically revealed a single nucleotide change (deletion, insertion, or substitution). Analyses of sequences, no template controls, and spurious alleles suggest no contamination during library preparation, pooling, and sequencing, but indicate that sequencing or PCR errors may have occurred due to DNA polymerase infidelities. Finally, we found utilizing Illumina’s FGx System at recommended conditions does not guarantee 100% outcomes for all samples tested, including the positive control, and required manual editing due to low read numbers and/or allele drop-in. These findings are important for progressing towards implementation of MPS in forensic DNA testing.

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

“…For example, not one of the positive controls, Illumina's 2800M, had all loci typed (Table ). Issues with this positive control were also reported previously . Positive controls are crucial as they validate the acceptability of a run.…”

“…Eye color predictions were also obtained using the Erasmus Medical Center's (EMC) web‐tool (https://hirisplex.erasmusmc.nl, ), and the Forensic Research/Reference on Genetics‐knowledge base (FROG‐kb) from Yale University (http://frog.med.yale.edu/FrogKB, ). These predictions for IrisPlex were obtained using all reads including those below 30, which were not typed by UAS, as it has been shown not including them may lead to incorrect genotypes . IrisPlex run through the EMC web‐tool based its predictions only on available genotypes.…”

“…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 .…”

The forensic landscape and the population genetic analyses of Hainan Li based on massively parallel sequencing DNA profiling

“…The ForenSeq™ DNA Signature Prep Kit, as the first commercial MPS-based and forensic related kit formally released in 2015, made full use of the MPS method's advantages by means of the simultaneous and compound amplification of either greater than 150 loci including various types of genetic markers. The large-scale performances and functional tests had been widely validated for the ForenSeq™ DNA Signature Prep Kit, the DNA Primer Set A (DPMA, 152 loci totally: 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 identity informative SNPs) and the DNA Primer Set B (in total 230 loci: DMPA plus 22 phenotypic informative SNPs and 56 biogeographical ancestry SNPs), which also include robustness, reproducibility, species specificity, consistency and sensitivity of detection [40][41][42][43][44][45][46][47]. In addition, the applicability of the DNA Signature Prep Kit had been evaluated and verified on challenging samples, DNA mixture, degradative DNA [48][49][50], formalin-fixed paraffin-embedded tissues [49,51], and remains of skeletons and bones [48,[52][53][54], to extend the applications for forensic sciences [55][56][57][58][59][60][61][62][63][64][65][66][67][68].…”

TranslucentID: Detecting Individuals with High Confidence in Saturated DNA SNP Mixtures

“…STR artifacts cannot be distinguished from alleles of low DNA contributor. Shifting from sizing STR alleles to HTS sequencing them [7][8][9] will enable resolution of different STR alleles with identical lengths, but PCR allele stutter artifacts will remain. Because of the importance of analyzing complex DNA forensic mixtures, multiple solutions have tackled DNA mixtures of more than two mixtures: ArmedXpert 10 …”