Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs.

The COVID-19 outbreak has represented a challenge for the international scientific community and particularly for forensic sciences. The lack of Coronavirus post-mortem testing led the National Institute of Toxicology and Forensic Sciences (INTCF) from Spain to verify the performance and utility of a quantitative reverse transcription PCR (RT-qPCR) clinical diagnosis protocol for SARS-CoV-2 detection (TaqPath™ COVID-19 CE-IVD RT-PCR Kit), to shed light on the cause of death (COD) in potentially COVID-19 cases in judicial autopsies. Two different RNA extraction methods were also tested (EZ1® DSP Virus Kit on the EZ1® Advanced XL robot versus MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit) regarding extraction efficiency, precision and contamination. RT-qPCR was evaluated for precision, specificity, limit of detection and concordance. Both the automated and the manual RNA extraction procedures showed good efficiency, but the automated virus extraction by bio-robot produced more reproducible results than the manual extraction. The SARS-CoV-2 RT-qPCR assay showed high sensitivity with a detection limit up to 10 copies/reaction and high specificity, as no cross-reactivity was detected between any of the 12 different RNA viruses tested, including three types of coronaviruses (SARS-CoV, NL63 and 229E). Reproducibility and repeatability of the studied method as well as concordance with other SARS-CoV-2 molecular detection protocols were also demonstrated.

The first GHEP-ISFG collaborative exercise on forensic applications of massively parallel sequencing.

One of the main goals of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. The GHEP-ISFG supports several Working Commissions which develop different scientific activities. One of them, the Working Commission on "Massively Parallel Sequencing (MPS): Forensic Applications", organized its first collaborative exercise on forensic applications of MPS technology in 2019. The aim of this exercise was to assess the concordance between the MPS results and those obtained with conventional technologies (capillary electrophoresis and Sanger sequencing), as well as to compare the results obtained within the different MPS platforms and/or the different kits/panels and analysis software packages (commercial and open-access) available on the market. The seven participating laboratories analyzed some samples of the annual GHEP-ISFG proficiency test (EIADN No. 27 (2019)), using Ion Torrent™ or MiSeq FGx® platforms. Six of them sent autosomal STR sequence data, five laboratories performed MPS analysis of individual identification SNPs, four laboratories reported MPS data of Y-chromosomal STRs, and X-chromosomal STRs, three laboratories performed MPS analysis of ancestry informative SNPs and phenotype informative SNPs, two labs performed MPS analysis of the mitochondrial DNA control region, and only one lab produced MPS data of lineage informative SNPs. Autosomal STR sequencing results were highly concordant to the consensus obtained by capillary electrophoresis in the EIADN No. 27 (2019) exercise. Furthermore, in general, a high level of concordance was observed between the results of the participating laboratories, regardless of the platform used. The main discordances were due to errors during the analysis process or from sequence data obtained with low depth of coverage. In this paper we highlight some issues that still arise, such as standardization of the nomenclature for STRs analyzed by sequencing with MPS, the universal uptake of a nomenclature framework by the analysis software, and well established validation and accreditation of the new MPS platforms for use in routine forensic case-work.

Microchip capillary electrophoresis protocol to evaluate quality and quantity of mtDNA amplified fragments for DNA sequencing in forensic genetics.

Here, we describe a microcapillary electrophoresis technique with application to the quantitative analysis of mtDNA hypervariable regions HVR1, HVR2, and HVR3 PCR amplicons previous to sequence analysis, which yields several important advantages compared to traditional separation and detection methods. Based on laser-induced fluorescence (LIF) detection, and performed in a microchip, this analysis system enables the handling of very small volumes via microchannels etched in the chip. Moreover it is faster than traditional methods; chip priming and sample loading are the only manual interventions, as the rest of the process is fully automated by software control: injection, electrophoretic separation, detection of the fluorescent signal, and calculation of both quantity and size. MtDNA amplicons are separated in microchannels with an effective length of 15 mm and detected by means of the fluorescence displayed by an intercalated dye. A software records the fluorescence and entails the data into size and concentration through the use of two internal standards and an external ladder of 11 fragments. The effectiveness of this procedure has been illustrated with a validation experiment carried out in our laboratory, in order to assess the detection limit of mtDNA sequencing by determining the minimal amount of PCR amplicon needed to edit a reproducible and high quality mtDNA sequence from complementary sequence data obtained using forward and reverse primers.