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Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples from altered human corpses using the MiSeq FGx system.

ABSTRACT: The proceeding developments in next generation sequencing (NGS) technologies enable increasing discrimination power for short tandem repeat (STR) analyses and provide new possibilities for human identification. Therefore, the growing relevance and demand in forensic casework display the need for reliable validation studies and experiences with challenging DNA samples. The presented validation of the MiSeq FGx system and the ForenSeq™ DNA Signature Prep Kit (1) investigated sensitivity, repeatability, reproducibility, concordance, pooling variations, DNA extraction method variances, DNA mixtures, degraded, and casework samples and (2) optimized the sequencing workflow for challenging samples from human corpses by testing additional PCR purification, pooling adjustments, and adapter volume reductions. Overall results indicate the system's reliability in concordance to traditional capillary electrophoresis (CE)-based genotyping and reproducibility of sequencing data. Genotyping success rates of 100% were obtained down to 62.5 pg DNA input concentrations. Autosomal STR (aSTR) profiles of artificially degraded samples revealed significantly lower numbers of locus and allelic dropouts than CE. However, it was observed that the system still exposed drawbacks when sequencing highly degraded and inhibited samples from human remains. Due to the lack of studies evaluating the sequencing success of samples from decomposed or skeletonised corpses, the presented optimisation studies provide valuable recommendations such as an additional PCR purification, an increase in library pooling volumes, and a reduction of adapter volumes for samples with concentrations ≥31.2 pg. Thus, this research highlights the importance of all-encompassing validation studies for implementing novel technologies in forensic casework and presents recommendations for challenging samples.

SUBMITTER: Senst A

PROVIDER: S-EPMC9313618 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:With the development and maturation of massively parallel sequencing (MPS) technology, the mitochondrial genome (mitogenome) sequencing is increasingly applied in the forensic field. In this study, we employed the strategy of short overlapping amplicons for the whole mitogenome, library preparation with tagmentation using the Nextera® XT DNA Library Preparation Kit, sequencing on the MiSeq FGxTM Forensic Genomics System and analyzing data using the mitochondrial(mtDNA) MSR Plug-in and the mtDNA Variant Analyzer. A total of 27 libraries and 56 libraries were sequenced in a run using MiSeq Reagent Kit v2 and v3, respectively. Results showed more than 1800 × of averaged depth of coverage (DoC) at each position. Concordant haplotypes of 9947 A and 2800 M were obtained at 32 variants. Cross-reactivity was observed with 1 ng primate DNA and 10 ng non-primate DNA but could be easily distinguished. Full and accurate variants were obtained from at least 50 pg input DNA and from minor contributors between 19:1 and 1:19 mixed ratios with known reference profiles. More than 86% variants were detected from ≥200-bp degraded samples but its haplotype was assigned to more ancestral haplogroup. Further, a total of 3 962 variants were observed at 613 nucleotide positions from 103 Xibe mitogenomes with 25:1 ratio of transitions to transversions. Two new transversions (C13735A and A14755C) and two tri-alleles at nps 9824 and 16092 were identified. There were 103 unique mitogenome haplotypes from 103 Chinese Xibe that were assigned to 79 haplogroups. Haplogroup D was the preponderant top-level haplogroup in Xibe followed by F, B, M, A, N, G, C, Z, Y, HV and J. Random match probability (RMP) and haplotype diversity (HD) of the whole mitogenome was calculated as 0.0097 and 1.0000, respectively. Compared with HVS-I only, RMP decreased 33.56%, while the number of haplotypes and HD increased 15.73% and 0.49%, respectively. Principal component analysis (PCA) showed that Xibe was clustered to East and Southeast Asian. As a whole, this MPS strategy is suitable for the whole mitogenome sequencing especially for degraded samples and can facilitate generating mitogenome data to support the routine application in forensic sciences. EMP00726 is the first whole mitogenome dataset from Xibe contributed to the EMPOP. Supplemental data for this article are available online at.

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Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples from altered human corpses using the MiSeq FGx system.

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