[Monitoring the spread of the SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) variants in the Moscow region using targeted high-throughput sequencing].

INTRODUCTION: Since the outbreak of the COVID-19 pandemic caused by SARS-CoV-2 novel coronavirus, the international community has been concerned about the emergence of mutations altering some biological properties of the pathogen like increasing its infectivity or virulence. Particularly, since the end of 2020, several variants of concern have been identified around the world, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). However, the existing mechanism of detecting important mutations are not always effective enough, since only a relatively small part of all pathogen samples can be examined by whole genome sequencing due to its high cost. MATERIAL AND METHODS: In this study, we have designed special primer panel and used it for targeted highthroughput sequencing of several significant S-gene (spike) regions of SARS-CoV-2. The Illumina platform averaged approximately 50,000 paired-end reads with a length of ≥150 bp per sample. This method was used to examine 579 random samples obtained from COVID-19 patients in Moscow and the Moscow region from February to June 2021. RESULTS: This study demonstrated the dynamics of distribution of several SARS-CoV-2 strains and its some single mutations. It was found that the Delta strain appeared in the region in May 2021, and became prevalent in June, partially displacing other strains. DISCUSSION: The obtained results provide an opportunity to assign the viral samples to one of the strains, including the previously mentioned in time- and cost-effective manner. The approach can be used for standardization of the procedure of searching for mutations in individual regions of the SARS-CoV-2 genome. It allows to get a more detailed data about the epidemiological situation in a region.

Metagenomic analysis of viruses in bat fecal samples from Moscow region reveals the whole genome sequences of Mastadenovirus and Alphacoronavirus

Due to the current epidemiological situation caused by the pandemic of COVID-19 the study of virus diversity in bats as a potential source of zoonotic diseases is crucially important. Our previous study based on genus specific PCR-using Coronaviridaespecific primers and following high throughput sequencing revealed the presence of several types of coronaviruses. In this study we performed the whole genome sequencing for the samples which were positive for Coronaviridae in order to get more information about viral communities in bat fecal samples. Fecal samples from bat species (Pippistrellus nathusii, Nyctalus noctula, Myotis brandtii, Myotis daubentonii) were collected in 2015 in the Moscow region and stored in RNA later. RNA extraction using Viral RNA Mini Kit (Qiaqen), library preparation using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) and HTS on Illumina Hiseq platform was performed. Metagenomic data was obtained using FastQC, trimmomatic 0.38 and SPAdes 3.13 for genome assembly, taxonomy of resulting contigs was identified by the BLASTn.For 8 bat fecal samples positive for Coronaviridae we performed the whole genome sequencing and metagenomic analysis. In one sample we detected the complete genome (37915 bp) with 74,66% nucleic identity to Mastadenovirus sp. isolate WA3301 from a Nyctalus noctula, in the other sample from Pippistrellus nathusii we found the complete genome sequence with 82% nucleotide identity to Bat alphacoronavirus isolate BtCoV/020-16/M.dau/FIN/2016 (28.245 bp).Whereas, as we've reported the presence of Alphacoronavirus before, this is the first time when we detect the whole genome of Mastadenovirus. The fact that the material was obtained from bats which nest next to human and domestic animals our findings might give better understanding the potential risk of the cross-species transmission and virus migration in bats from Moscow Region.

Effect of oropharyngeal swab quality on the success of NGS library preparation

SARS-CoV-2 genomes rapidly change due to mutations, and thus the fast evolution of this virus has been observed worldwide. As is known, the majority of the identified mutations do not influence a significant effect on the spread. While some mutations or combinations can provide the virus advantages because some strains spread quickly around local populations. We had tested 477 oropharyngeal swabs from patients from different regions of Russia with diagnosis COVID-19 to perform a whole-genome sequencing to detect new variants of SARS-CoV-2. We were faced with the problem of low-quality samples. It had led to the deterioration of amplification. It's a significant moment to perform screening tests. Methods: Previously designed primers panel [1] was used for SARS-CoV-2 whole genome amplification. The PCR products ranging from 1757 to 2054 bp were mixed, purified. Libraries were constructed using Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096). Sequencing was performed as described in [1]. The consensus sequence was submitted to GISAID database (hCoV-19/Russia/CRIE). Results: We completely amplified and whole-genome sequenced the 173 of SARS-CoV-2 genomes from 477 samples. The success of genome fragments amplification varied from 76% to zero when we used samples obtained from different sources (from different clinics). We suppose that this dramatic difference could be explained by the composition of transportation buffers that are purchased by different clinics for routine diagnostics of COVID-19 by RTPCR methods. Our results demonstrated that the success of amplification does not depend on the storage and transportation time.

Genetic variability of SARS-CoV-2 in biological samples from patients in Moscow

Currently, a lot of attention is given to SARS-CoV-2 subpopulations and their coexistence with different genomic variants within the same patient. In this study, we performed next-generation whole-genome sequencing and assembly of viruses from samples representing swabs or autopsy specimens obtained from patients diagnosed with СOVID-19, which were initially confirmed by the real-time polymerase chain reaction (Ct = 10.4-19.8). Samples were prepared for sequencing by using the SCV-2000bp protocol. The obtained data were checked for presence of more than one SARS-CoV-2 genetic variants in a sample. Variants of nucleotide substitutions, coverage for each variant, and location of the variable position in the reference genome were detected with tools incorporated in the CLC Genomics Workbench program. In our search for variable nucleotide positions, we assumed that the sample had two genetic variants (not more);the threshold value ≥ 90% was set for probability of the identified variant. Variants represented by less than 20% of the reads in the total coverage were not taken into consideration. The obtained results showed that 5 samples had variability, i.e. they had several genetic variants of SARS-CoV-2. In 4 samples, both of the detected genomic variants differed only in one nucleotide position. The fifth sample demonstrated more substantial differences: a total of 3 variable positions and one three-nucleotide deletion. Our study shows that different genetic variants of SARS-CoV-2 can coexist within the same patient. Сосуществование субпопуляций SARS-CoV-2 с различными вариантами генома внутри организма одного пациента - один из обсуждаемых в настоящее время феноменов. В данной работе мы провели высокопроизводительное секвенирование и сборку полных геномов вирусов из образцов, которые представляли собой мазки или аутопсийный материал от пациентов с диагнозом СOVID-19, предварительно подтвержденным методом полимеразной цепной реакции в реальном времени (Ct = 10,4-19,8). Подготовку образцов к секвенированию проводили с помощью протокола SCV-2000bp. Полученные данные проверяли на присутствие в образце более чем одного генетического варианта SARS-CoV-2. Варианты нуклеотидных замен, покрытие для каждого варианта, а также координаты вариабельной позиции в референсном геноме определяли с помощью инструментов программы «CLC Genomics Workbench». При поиске вариабельных нуклеотидных позиций исходили из предположения, что в образце имеются 2 генетических варианта (не более), для вероятности определяемого варианта использовали пороговое значение ≥ 90%. Также игнорировали варианты, которые были представлены менее чем 20% прочтений от общего покрытия. Полученные результаты показали, что в 5 образцах имеется вариабельность, т.е. содержится несколько генетических вариантов SARS-CoV-2. В 4 образцах оба найденных варианта геномов различались лишь в одной нуклеотидной позиции. В пятом образце были найдены более существенные различия: сразу 3 вариабельных позиции и одна делеция длиной в 3 нуклеотида. Наше исследование показывает возможность сосуществования различных генетических вариантов SARS-CoV-2 в организме пациента.