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.

Streamlining the decision-making process for international DNA kinship matching using Worldwide allele frequencies and tailored cutoff log10LR thresholds.

The identification of human remains belonging to missing persons is one of the main challenges for forensic genetics. Although other means of identification can be applied to missing person investigations, DNA is often extremely valuable to further support or refute potential associations. When reference DNA samples cannot be collected from personal items belonging to a missing person, a direct DNA identification cannot be carried out. However, identifications can be made indirectly using DNA from the missing person's relatives. The ranking of likelihood ratio (LR) values, which measure the fit of a missing person for any given pedigree, is often the first step in selecting candidates in a DNA database. Although implementing DNA kinship matching in a national environment is feasible, many challenges need to be resolved before applying this method to an international configuration. In this study, we present an innovative and intuitive method to perform international DNA kinship matching and facilitate the comparison of DNA profiles when the ancestry is unknown or unsure and/or when different marker sets are used. This straightforward method, which is based on calculations performed with the DNA matching software BONAPARTE, Worldwide allele frequencies and tailored cutoff log10LR thresholds, allows for the classification of potential candidates according to the strength of the DNA evidence and the predicted proportion of adventitious matches. This is a powerful method for streamlining the decision-making process in missing person investigations and DVI processes, especially when there are low numbers of overlapping typed STRs. Intuitive interpretation tables and a decision tree will help strengthen international data comparison for the identification of reported missing individuals discovered outside their national borders.

Multi-laboratory validation of DNAxs including the statistical library DNAStatistX.

This study describes a multi-laboratory validation of DNAxs, a DNA eXpert System for the data management and probabilistic interpretation of DNA profiles [1], and its statistical library DNAStatistX to which, besides the organising laboratory, four laboratories participated. The software was modified to read multiple data formats and the study was performed prior to the release of the software to the forensic community. The first exercise explored all main functionalities of DNAxs with feedback on user-friendliness, installation and general performance. Next, every laboratory performed likelihood ratio (LR) calculations using their own dataset and a dataset provided by the organising laboratory. The organising laboratory performed LR calculations using all datasets. The datasets were generated with different STR typing kits or analysis systems and consisted of samples varying in DNA amounts, mixture ratios, number of contributors and drop-out level. Hypothesis sets had the correct, under- and over-assigned number of contributors and true and false donors as person of interest. When comparing the results between laboratories, the LRs were foremost within one unit on log10 scale. The few LR results that deviated more had differences for the parameters estimated by the optimizer within DNAStatistX. Some of these were indicated by failed iteration results, others by a failed model validation, since unrealistic hypotheses were included. When these results that do not meet the quality criteria were excluded, as is in accordance with interpretation guidelines, none of the analyses in the different laboratories yielded a different statement in the casework report. Nonetheless, changes in software parameters were sought that minimized differences in outcomes, which made the DNAStatistX module more robust. Overall, the software was found intuitive, user-friendly and valid for use in multiple laboratories.

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.

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

We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer's recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.

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.

[The future of forensic DNA analysis for criminal justice]. / Les nouvelles technologies d'analyses ADN au service des enquêtes judiciaires.

In the criminal framework, the analysis of approximately 20 DNA microsatellites enables the establishment of a genetic profile with a high statistical power of discrimination. This technique gives us the possibility to establish or exclude a match between a biological trace detected at a crime scene and a suspect whose DNA was collected via an oral swab. However, conventional techniques do tend to complexify the interpretation of complex DNA samples, such as degraded DNA and mixture DNA. The aim of this review is to highlight the powerness of new forensic DNA methods (including high-throughput sequencing or single-cell sequencing) to facilitate the interpretation of the expert with full compliance with existing french legislation.

New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats.

Myotonic Dystrophy type I (DM1) is caused by an abnormal expansion of CTG triplets in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene, leading to the aggregation of the mutant transcript in nuclear RNA foci. The expanded mutant transcript promotes the sequestration of the MBNL1 splicing factor, resulting in the misregulation of a subset of alternative splicing events. In this study, we identify the DEAD-box RNA helicase p68 (DDX5) in complexes assembled onto in vitro-transcribed CUG repeats. We showed that p68 colocalized with RNA foci in cells expressing the 3'UTR of the DMPK gene containing expanded CTG repeats. We found that p68 increased MBNL1 binding onto pathological repeats and the stem-loop structure regulatory element within the cardiac Troponin T (TNNT2) pre-mRNA, splicing of which is misregulated in DM1. Mutations in the helicase core of p68 prevented both the stimulatory effect of the protein on MBNL1 binding and the colocalization of p68 with CUG repeats, suggesting that remodeling of RNA secondary structure by p68 facilitates MBNL1 binding. We also found that the competence of p68 for regulating TNNT2 exon 5 inclusion depended on the integrity of MBNL1 binding sites. We propose that p68 acts as a modifier of MBNL1 activity on splicing targets and pathogenic RNA.