The goal of this part of the study was to optimize the sequencing procedure for 16 human genes and their regulatory regions that might be associated with differential immunological response to COVID-19. The study was performed on 60 COVID-19 patients from the General Hospital of Tešanj, Bosnia and Herzegovina, categorized into three groups of mild, moderate, and severe clinical manifestation, based on the diagnosis by the residential physician. Target coding sequences and their regulatory regions were amplified for the following genes: HLA-A, HLA-B, HLA-C, ACE2, IL-6, IL-4, TMPRSS2, IFITM3, IL-12, RIG-I/DDX58, IRF-7, IRF-9, IL-1B, IL-1A, CD55, and TNF-α. DNA was isolated from the whole blood samples stored at -20°C for six months using QIAamp® DNA Mini Kit according to manufacturer’s instructions. Since NGS analysis of target genomic regions was performed on the Ion Torrent GeneStudio™ S5 platforms, libraries were prepared using Ion AmpliSeq™ Library Kit Plus according to manufacturer’s instructions in a protocol optimized for low-quality DNA. Due to dissatisfactory sequencing results, further protocol optimization steps were employed through separating two primer pools, increasing the number of PCR cycles, and decreasing the annealing temperature for the primer pool which showed poorer amplification results. In the end, 36 samples produced optimal results, while the remaining 24 samples will be re-sequenced following repeated sample collection and DNA isolation, accompanied by additional protocol modifications.
Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.
IntroductionSerological detection of SARS-CoV-2-specific IgG and IgM antibodies is becoming increasingly important in management of COVID-19 pandemic.Material and methodsWe are reporting the first results of COVID-19 serological testing in Bosnia and Herzegovina on 2,841 samples collected and analyzed in two medical institutions in Sarajevo. Antibody detection was performed using commercially available kits.ResultsIn the first cohort, 43 IgM-positive/IgG-negative and 16 IgM-positive/IgG-positive individuals were detected, corresponding to 3.41% of participants having developed antibodies. In the second cohort, 4.28% participants were found to be IgM-negative/IgG-positive.ConclusionsOur results suggest need for population-wide serological surveying in B&H.
COVID-19, caused by the SARS-CoV-2 virus, has been a major focus of scientific research since late 2019 and early 2020. Due to its enormous societal, economic, and clinical impact worldwide, research efforts aimed, among other questions, to address the effect of host genetics in susceptibility and severity of COVID-19. In this research, we performed next-generation sequencing of coding and regulatory regions of 16 selected human genes, involved in the maintenance of the immune system or encoding the receptors for viral entry into the host cells, in a subset of 60 COVID-19 patients from the General Hospital Tešanj, Bosnia and Herzegovina, classified into three groups of patients with clinical conditions of different severity (“mild”, “moderate”, and “severe” clinical groups). In accordance with previous studies, we found out that the male sex and older age are risk factors for severe clinical picture. We identified 13 variants on seven genes (CD55, IL1B, IL4, IRF7, DDX58, TMPRSS2, and ACE2) with potential functional significance, either as genetic markers of modulated susceptibility to SARS-CoV-2 infection or as modifiers of the course of infection in terms of predicted symptom severity. Our results include variants reported for the first time as potentially associated with COVID-19. Future studies on larger patient cohorts, focused on candidate genes and/or candidate genetic variants, have a potential to answer a range of open questions regarding the effect of host (human) genetic makeup on the expected outcome of COVID-19.
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