key: cord-0840797-q7tsfzhr
authors: Biazzo, Manuele; Madeddu, Silvia; Elnifro, Elfath; Sultana, Tessabella; Muscat, Josie; Scerri, Christian A.; Santoro, Francesco; Pinzauti, David
title: Genome Sequences of 10 SARS-CoV-2 Viral Strains Obtained by Nanopore Sequencing of Nasopharyngeal Swabs in Malta
date: 2021-01-28
journal: Microbiol Resour Announc
DOI: 10.1128/mra.01375-20
sha: f52ad761e896db69fec056a9d19bee44de95e613
doc_id: 840797
cord_uid: q7tsfzhr

The genome sequences of 10 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains from Sliema, Malta, were obtained by Nanopore sequencing using the amplicon sequencing approach developed by the Artic Network. The assembled genomes were analyzed with Pangolin software and assigned to the B.1 lineage, which is widely circulating in Europe.

W e present the genome sequences of 10 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; genus Betacoronavirus, family Coronaviridae) viral strains retrieved from Sliema, Malta. Because of a relatively high incidence of SARS-CoV-2 infection in Malta, we decided to sequence these viral strains to contribute to the local and global epidemiology of SARS-CoV-2. Rapid sequencing is important for surveillance of circulating viral strains and for prompt contact tracing (1) . Viral RNA was obtained from nasopharyngeal swabs collected at the St. James Hospital (Sliema, Malta) in August and September 2020 ( Table 1 ). The research described in this work was performed in adherence to the Declaration of Helsinki; no specific authorization was issued by the University of Malta Institutional Review Board (IRB), since samples were anonymized and no human data were used. Swabs were placed in 3 ml ImproViral viral preservative medium (VPM) (Improve Medical). Viral RNA was extracted with a MagCore viral nucleic acid extraction kit (RBC Bioscience; catalog number MVN400-04) using a MagCore Plus II automated nucleic acid extractor (RBC Bioscience) and was then tested by quantitative PCR (qPCR) to assess the presence of SARS-CoV-2 RNA using the GrandPerformance SARS-CoV-2 kit (TATAA Biocenter, Sweden) targeting the RNA-dependent RNA polymerase gene. Viral RNA from qPCR-positive samples was sequenced using the multiplex PCR amplicon sequencing approach developed by the ARTIC Network (https://www.protocols.io/view/ncov-2019-sequencing-protocol-v2 -bdp7i5rn?version_warning=no&step=16.3). Primers for 98 overlapping amplicons were used in two multiplex PCRs to amplify the whole viral genome, except for the conserved 59 and 39 untranscribed regions (UTRs). The sequencing library was prepared using the native barcode kit EXP-NBD104 from Oxford Nanopore Technologies (ONT) and sequenced on an R9.4 flow cell using a MinION MK1B device (ONT). The sequencing run was managed with MinKNOW v19.12.5, disabling live base calling. Raw Nanopore reads were base called using the GridION Guppy module v4.0.11, enabling the high-accuracy mode. Base-called reads were analyzed following the ARTIC Network pipeline (https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html). All tools were run using default parameters unless otherwise specified. In total, 2,645,399 reads were obtained (mean, 264,540; range, 73,135 to 468,322 per sample). Table 1 summarizes the information for each sample, including genome coverage since BLAST analysis of the Betacoronavirus NCBI resource indicated high similarity with the Sliema genomes. Using the cat command from the Linux environment, representative genomes and Sliema samples were merged and then aligned to the reference Wuhan Hu-1 genome using the MAFFT algorithm v7.471 (5) . The resulting alignment was used by IQ-TREE to reconstruct the phylogenetic tree with an ultrafast bootstrap value of 1,500 (6) . The tree file was finally visualized using the Interactive Tree of Life (iTOL) Web tool (7) (Fig. 1) . The Malta genomes all belonged to a separate subcluster, indicating that viral strains circulating in Malta in September 2020 were genetically homogenous. Genomic epidemiology is essential for monitoring the emergence and spread of SARS-CoV-2 variants. Data availability. The Nanopore reads and genome sequences of the SARS-CoV-2 Malta/Sliema isolates are publicly available. The Sequence Read Archive (SRA) and GenBank accession numbers are listed in Table 1 .

Rapid implementation of SARS-CoV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study

Using Tablet for visual exploration of second-generation sequencing data

A dynamic nomenclature proposal for SARS-CoV-2 to assist genomic epidemiology

IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies

MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform

UFBoot2: improving the ultrafast bootstrap approximation

Interactive Tree Of Life (iTOL) v4: recent updates and new developments

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.