engleski

COVID-19 pandemic from the forensic standpoint: A platform against future biological threats

For almost a year and a half, the world has been struggling with challenges brought upon global society by COVID-19 pandemic. The effort to adapt to private and professional life conditions became an everyday routine for millions of individuals, businesses and social entities alike. We as governmental forensic DNA laboratory attempting to contribute to the COVID-19 response, have also experienced disruptions in forensic research activities, both in terms of human potential and infrastructure. Having recognized our competences, academic partners included us in the nationally funded scientific project aiming at SARS-CoV-2 and COVID-19 host genetic sequencing [1]. Here we describe the extent of our contribution based on the internally validated protocol for massively parallel sequencing (MPS) of the whole mitochondrial genomes [2]. Additionally, we report improvements of expertise on virus MPS by performing SARS-CoV-2 sequencing. Besides the evident social benefit from the collaboration of the national forensics with the academia as a response to the immediate pandemic threat, this collaborative model opens up new aspects of future forensic research and development in the area of forensic microbiology. MPS workflow of the Forensic Science Centre “Ivan Vučetić” Research DNA Laboratory is based on the Illumina® MiSeqFGx® instrument, for the whole mitochondrial DNA (mtDNA) sequencing from blood samples of COVID-19 patients (MiSeq® Reagent Kit v2 300cyc) and SARS-CoV-2 sequencing from patients’ corresponding nasopharyngeal swabs (MiSeq® Reagent Kit v3 500cyc). Samples were collected, viral RNA was extracted (SaMagTM ViralNA Extraction Kit, Sacace Biotechnologies) and quantified (SARS- CoV-2 Real-TM, Sacace Biotechnologies) at the University Hospital Dubrava. Viral RNA was submitted to cDNA conversion (Quanti Tect® Reverse Transcription Kit, Qiagen) and library preparation (NEBNext® UltraTM II DNA Library Prep Kit, New England Biolabs) at the Ruđer Bošković Institute, while the entire mtDNA workflow was conducted at the Forensic Science Centre “Ivan Vučetić” using Illumina® NexteraXT Library Prep Kit, as described previously [2]. Raw sequencing data were processed at the University of Zagreb, Faculty of Science. Sequences were mapped to reference genomes (either SARS-Cov-2 or human mtDNA) and variant calling was performed to determine viral mutations and mitochondrial haplogroups. Ongoing project activities have so far resulted in optimization of the complex workflow, involving many researchers and institutions. Best results were obtained when nucleic acid samples were stored for less than 30 days after isolation, and from individuals with the SARS- CoV-2 detection real-time PCR Ct values below 30. The molarity of viral sequencing libraries should exceed 5 nM. Blood samples that were already used for the biochemical analyses are not compatible with mtDNA heteroplasmy analysis due to frequent cross-contamination, mandating the need for intact samples. For optimal sequencing cluster density, the molarity of SARS-CoV-2 libraries was increased from 12 to 20 pM. For higher viral sequence coverage, PhiX control spike-in was decreased from 10% to 5%. Detected viral variants and their relative amounts followed patterns observed in neighboring countries and the rest of Europe (sequences available from GISAID and lineage assigned using PANGOLIN). Mitochondrial sequencing confirmed overall haplogroup distributions similar to that of the general population, however with interestingly different haplogroup ratios between patient groups with mild and severe symptoms (fivefold higher proportion of macrohaplogroup K in patients with mild symptoms). Potential protective effect of any mitochondrial haplogroup should be further investigated in expanded patient pool. Experience gained through this project is of huge benefit to the entire community, as multi- institutional effort in characterizing SARS- CoV-2 might be reflected in different forensic scenarios involving accidental or intentional biological agent release. We will therefore continue to strengthen and improve upon the established collaborative network and procedures beyond the current COVID-19 crisis, in order to minimize the harm from future biological incidents. [1] Grant ID: IP-CORONA-2020-04-2084, funded by Croatian National Science Foundation [2] Sukser V, Rokić F, Barbarić L and Korolija M: Assessment of Illumina® Human mtDNA Genome assay: workflow evaluation with development of analysis and interpretation guidelines. International Journal of Legal Medicine 135 (2021) 1161-1178 (doi: 10.1007/s00414-021- 02508-z)

COVID-19 pandemic ; SARS-Cov-2 sequencing ; mtDNA sequencing ; biodefence

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