SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system

Raffaella Pasquale,Francesca Bergantino,Luca De Sabato,Mariano Fiorenza,Daniela Terracciano,Michele Cennamo,Gerardo Botti,Cristin Roma,Gabriele Vaccari,Giuseppe Portella,Ernesta Cavalcanti,Anna Maria Rachiglio,Nicola Normanno

doi:10.1186/s12967-021-02912-4

Abstract

BackgroundSince the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database. Sequencing of the SARS-CoV-2 genome might allow identification of variants with increased contagiousness, different clinical patterns and/or different response to vaccines. A highly automated next generation sequencing (NGS)-based method might facilitate an active genomic surveillance of the virus.MethodsRNA was extracted from 27 nasopharyngeal swabs obtained from citizens of the Italian Campania region in March–April 2020 who tested positive for SARS-CoV-2. Following viral RNA quantification, sequencing was performed using the Ion AmpliSeq SARS-CoV-2 Research Panel on the Genexus Integrated Sequencer, an automated technology for library preparation and sequencing. The SARS-CoV-2 complete genomes were built using the pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) and analysed by IQ-TREE software.ResultsThe complete genome (100%) of SARS-CoV-2 was successfully obtained for 21/27 samples. In particular, the complete genome was fully sequenced for all 15 samples with high viral titer (> 200 copies/µl), for the two samples with a viral genome copy number < 200 but greater than 20, and for 4/10 samples with a viral load < 20 viral copies. The complete genome sequences classified into the B.1 and B.1.1 SARS-CoV-2 lineages. In comparison to the reference strain Wuhan-Hu-1, 48 total nucleotide variants were observed with 26 non-synonymous substitutions, 18 synonymous and 4 reported in untranslated regions (UTRs). Ten of the 26 non-synonymous variants were observed in ORF1ab, 7 in S, 1 in ORF3a, 2 in M and 6 in N genes.ConclusionsThe Genexus system resulted successful for SARS-CoV-2 complete genome sequencing, also in cases with low viral copies. The use of this highly automated system might facilitate the standardization of SARS-CoV-2 sequencing protocols and make faster the identification of novel variants during the pandemic.

Highlights

Since the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database
Coronavirus disease-19 (COVID-19), declared as pandemic on March 2020 by WHO, is an infectious disease caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2)
The analysis performed with the TaqPathTM COVID19 RT-PCR test found that nine samples had a viral load > 1000 copies/μl, six had a load between 200 and 1000 copies/μl and two samples carried < 200 copies/μl of the SARS-CoV-2 viral genome

Summary

Introduction

Since the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database. A highly automated generation sequencing (NGS)-based method might facilitate an active genomic surveillance of the virus. Coronavirus disease-19 (COVID-19), declared as pandemic on March 2020 by WHO, is an infectious disease caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). As of December 2019, SARS-CoV-2 infected more than 114 million individuals worldwide, causing more than 2.5 million deaths [4]. The first wave of infection mainly affected the Northern Italian regions, causing thousands of deaths, especially among the most fragile individuals [5]. As of March 1, 2021, Italy has been affected by 2,955,434 cases and 98,288 deaths (www.salute.gov.it)

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