Evaluation of the VISAGE basic tool for appearance and ancestry inference using ForenSeq® chemistry on the MiSeq FGx® system.

The possibility of providing investigative leads when conventional DNA identification methods fail to solve a case can be of extreme relevance to law enforcement. Therefore, the forensic genetics community has focused research towards the broadened use of DNA, particularly for prediction of appearance traits, bio-geographical ancestry and age. The VISible Attributes through GEnomics (VISAGE) Consortium expanded the use of DNA phenotyping by developing new molecular and statistical tools for appearance, age and ancestry prediction. The VISAGE basic tool for appearance (EVC) and ancestry (BGA) prediction was initially developed using Ampliseq chemistry, but here is being evaluated using ForenSeq chemistry. The VISAGE basic tool offers a total of 41 EVC and 115 BGA SNPs and thus provides more predictions, i.e., skin color, than achieved with the ForenSeq DNA Signature Prep kit that is based on 24 EVC and 56 BGA SNPs. Five VISAGE laboratories participated in collaborative experiments to provide foreground for developmental validation of the assay. Assessment of assay performance and quality metrics, reproducibility, sensitivity, inhibitor tolerance and species specificity are described. Furthermore, the assay was tested using challenging samples such as mock casework samples and artificially degraded DNA. Two different analysis strategies were applied for this study and output on genotype calls and read depth was compared. Overall, inter-laboratory, inter-method and concordance with publicly available data were analysed and compared. Finally, the results showed a reliable and robust tool, which can be easily applied for laboratories already using a MiSeq FGx with ForenSeq reagents.

Sequenced-based French population data from 169 unrelated individuals with Verogen's ForenSeq DNA signature prep kit.

Massively Parallel Sequencing (MPS) applied to forensic genetics allows the simultaneous analysis of hundreds of genetic markers and the access to full amplicon sequences which help to increase available allele diversity. Meanwhile, sequence variation within the repeat regions represents the majority of the allele diversity, flanking regions adjacent to the repeat core provide an additional degree of variation. The forensic genetics community needs access to population data, from relevant parts of the world that contain this new sequence diversity in order to perform statistical calculations. In this study, we report sequence-based Short Tandem Repeat (STR) and identity Single Nucleotide Polymorphism (iSNPs) allele data for 169 French individuals across 58 STRs and 92 SNPs included in the Verogen ForenSeq DNA Signature Prep kit. 42 STRs out of 58 showed an increased number of alleles due to sequence variation in the repeat motif and/or the flanking regions. D9S1122 showed the largest overall gain with an increase of observed heterozygosities of almost 25 %. The combined match probability combining 27 autosomal STRs and 91 identity SNPs was 1.11E-69. Sequence-based allele frequencies included in this publication will help forensic laboratories to increase the power of discrimination for identification, kinship analysis and mixture interpretation.

Automation and developmental validation of the ForenSeq™ DNA Signature Preparation kit for high-throughput analysis in forensic laboratories.

Massively parallel sequencing (MPS) applications in forensic science highlight the advantages of this technique compared to capillary electrophoresis (CE). The multiplexing of a wide range of genetic markers and access to the full amplicon sequence, allowing the detection of isoalleles, make it a very promising tool which could be applied to the most challenging casework DNA samples. However, the complexity of the manual library preparation protocol, potential DNA contamination and sample tracking issues are the main reasons why forensic scientists still hesitate to implement MPS analytical workflows in their laboratory. Here, we present the automation of all library preparation steps for up to 96 samples using the Verogen's ForenSeq™ DNA Signature Preparation kit. This automated protocol, developed on a Hamilton ID STARlet robotic platform, is designed to allow the combined sequencing of rich and poor DNA samples thanks to a final step which adjusts normalized library pooling volume to guarantee a uniform depth of coverage across all samples. Our study includes tests of concordance, repeatability, reproducibility and sensitivity (1000 pg, 700 pg, 500 pg, 250 pg, 100 pg and 50 pg). Sequencing results obtained with the automated protocol were found to be concordant with previous validation studies using the manual protocol in terms of depth of coverage and allele coverage ratio. The results of this study will assist forensic laboratories seeking to acquire a plug and play solution to optimize the processing and analysis of casework samples with MPS.