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

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

Research output: Contribution to journalArticlepeer-review

Abstract

Background: 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. 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. Methods: RNA 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. Results: The 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. Conclusions: The 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.

Original languageEnglish
Article number246
JournalJournal of Translational Medicine
Volume19
Issue number1
DOIs
Publication statusPublished - Dec 2021

Keywords

  • Campania region
  • Covid-19
  • Next generation sequencing
  • SARS-CoV-2 genome

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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