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Next generation sequencing (NGS) technologies are going to play a major role in forensic genomics in the next few years. A new NGS platform, the MiSeq FGx™ Forensic Genomics System (Illumina Inc., San Diego) was launched in January 2015 as the first fully validated sequencing system specifically designed for forensic genomics applications. This new system allows simultaneous, high resolution sequencing of 152 identity informative markers (including 27 autosomal STRs, 7 X-chromosomal, 24 Y-chromosomal haplotype markers and 94 SNPs) plus 56 ancestry and 22 phenotypic informative SNPs.
In order to assess the flexibility of MiSeq FGx™ Forensic Genomics System for STR genotyping, a concordance study on samples previously typed using the ABI 3130 Genetic Analyzer with GeneMapper ID-X v1.2 software was performed. Results were compared for 22 autosomal and 27 gonosomal STRs previously analysed and subsequently sequenced using the Forenseq DNA Signature Prep kit and analyzed with the Forenseq Universal Analysis software. Here we present the results of a preliminary study within our laboratory and discuss the likely expectation that the MiSeq FGx™ Sequencing System technology could provide more powerful analytical tools for the forensic genomics field.
]. The new NGS platform from Illumina, MiSeq FGx™ Forensic Genomics System is the first fully validated sequencing system specifically designed for forensic genetics applications. This system is provided with the Forenseq™ DNA Signature Prep kit (Illumina), allowing simultaneous, high resolution sequencing of 230 loci, including 152 identity-informative markers (27 autosomal, 7 X-chromosomal, 24 Y-chromosomal STRs and 94 identity-informative (IISNPs) SNPs), 56 ancestry (AISNPs) and 22 phenotype-informative SNPs (PISNPs).
In this study, we analyzed forensic samples from our database to provide a preliminary evaluation of this NGS platform and assess genotyping concordance with the ABI 3130 sequencer.
2. Materials and methods
A total of 78 DNA samples obtained from individual involved in paternity testing (n = 64) and crime caseworks (n = 14) were selected. In this last group, 5 DNA samples represented traces collected at the crime scene and included two DNA mixtures, while, the 9 remaining were collected from skeletal muscles, nails and teeth. All samples were previously typed for 22 autosomal STRs (Promega), 12 X-(Qiagen) and 23Y-chromosomal STRs (Promega) using an ABI Prism 3130 with GeneMapper ID-X v1.2 software. DNA samples were quantified using the Qubit® dsDNA HS Assay with the Qubit Fluorimeter (Qiagen) and subsequently normalized to a concentration of 0.2 ng/μl. DNA libraries were generated with the Forenseq™ DNA Signature Prep kit (Illumina) [
] using the two panels of markers provided, named DNA Primer Mix A (59 STRs + 94 IISNPs) and B (including all markers of Panel A + 56 AISNPs and 22 PISNPs), according to the manufacturer’s instructions. Sequencing data were analyzed by means of the Forenseq™ Universal Analysis Software (Illumina). Quality metrics of the runs were evaluated in crime casework and paternity samples by considering the number of cycles completed and the number of sequencing reads per sample/per marker/per allele.
3. Results and discussion
As expected, due to the good quality of DNA, sequence coverage (expressed as the number of sequencing reads per sample, r) was, on average, higher for paternity testing (r = 182000) than crime casework samples (r = 131000). For the latter, 25% of DNA samples displayed a mean coverage of 31,605 reads, not sufficient to overcome the minimal signal intensity (85,000 reads), required from the Forenseq™ Universal Analysis Software (Illumina) for genotyping purposes. However, this aspect seems to not affect significantly the resulting LR/RMP values giving that the average number of STR and SNP markers that were correctly called was of 44/59 and 62/94, respectively. In the remaining 75% of crime casework samples, the average number of STRs and SNPs that could be reliably genotyped was 58/59 and 91/94, respectively.
All 78 samples tested gave full genotyping concordance at STR markers shared between the Forenseq™ DNA Signature Prep kit (Illumina) and PowerPlex Fusion, PowerPlex Y23 (Promega) and Investigator Argus X-12 (Qiagen) kits (Fig. 1). Despite a complete identity between STR genotyping results of the two platforms, NGS sequencing highlighted additional relevant sequence information for several STR alleles. These variants, revealed also in the 2800 M cell line DNA (positive control) at the D9S1122 locus, could be extremely useful in deconvoluting DNA mixtures recovered at the crime scene, especially when the contributors are close relatives or autochthonous subjects. The two DNA mixtures analyzed by the MiSeq FGx revealed intra-repeat STR variations in an apparently identical allele, shared by the victim and the perpetrator (Fig. 2).
The results obtained from this first evaluation and concordance study, confirmed the genotyping accuracy of Next Generation Sequencing by means of MiSeq FGx System and overcome the CE technology on sequencing resolution. Surely this new platform will provide additional discrimination power to STRs routinely used in forensic genetics considering its ability in revealing intra-repeat sequence variants. The possibility of detect 59 autosomal/gonosomal STRs together with 94 SNPs in a single sequencing run, allows a better optimization of DNA recovered from a crime scene, without forgetting the additional information provided by ancestry and phenotype prediction.
Conflict of interest
The authors have disclosed no conflict of interest.
This work was supported by a grant from the “Donazione M. Cherubini and A. Loro”
Next generation sequencing and its applications in forensic genetics.