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.
Highlights
Parallel Sequencing (MPS) has several advantages over conventional techniques in clinical and forensic applications as Massively Parallel Sequencing (MPS) can be more cost effective and generates more informative data [1,2,3,4]
Some short tandem repeats (STRs) and identity SNPs (iSNPs) were missed in Expt
This study focused on a novel method for forensic DNA analysis, massively parallel sequencing (MPS)
Summary
Parallel Sequencing (MPS) has several advantages over conventional techniques in clinical and forensic applications as MPS can be more cost effective and generates more informative data [1,2,3,4]. Before incorporating new technologies into routine laboratory operations they must be evaluated for performance This may include tests for accuracy, robustness, precision, repeatability, reproducibility, analytical sensitivity, and specificity [1, 6, 9]. Several manufacturers have designed MPS kits for forensic testing that include the Combined DNA Index System (CODIS) STRs that are used in the United States for criminal investigations These kits often include additional autosomal, X, and Y chromosome STRs, as well as identity, ancestry, and phenotypic SNPs (iSNPs, aSNPs, pSNPs). The latter, in combination with complete MPS mitochondrial sequencing kits, could add important information for the identification of missing persons when only partial or skeletal remains are available. These kits were designed to be utilized with the major MPS sequencing platforms currently in use: Illumina’s MiSeq (San Diego, CA) and Thermo Fisher’s Ion PGM and S5 (Waltham, MA)
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