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SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system.
Rachiglio, Anna Maria; De Sabato, Luca; Roma, Cristin; Cennamo, Michele; Fiorenza, Mariano; Terracciano, Daniela; Pasquale, Raffaella; Bergantino, Francesca; Cavalcanti, Ernesta; Botti, Gerardo; Vaccari, Gabriele; Portella, Giuseppe; Normanno, Nicola.
  • Rachiglio AM; Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori "Fondazione Giovanni Pascale", IRCCS, Via Mariano Semmola, 80131, Napoli, Italy.
  • De Sabato L; ISS-Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, 00161, Rome, Italy.
  • Roma C; Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori "Fondazione Giovanni Pascale", IRCCS, Via Mariano Semmola, 80131, Napoli, Italy.
  • Cennamo M; Dipartimento Scienze Mediche Traslazionali, Università di Napoli "Federico II", 80131, Napoli, Italy.
  • Fiorenza M; Dipartimento Scienze Mediche Traslazionali, Università di Napoli "Federico II", 80131, Napoli, Italy.
  • Terracciano D; Dipartimento Scienze Mediche Traslazionali, Università di Napoli "Federico II", 80131, Napoli, Italy.
  • Pasquale R; Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori "Fondazione Giovanni Pascale", IRCCS, Via Mariano Semmola, 80131, Napoli, Italy.
  • Bergantino F; Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori "Fondazione Giovanni Pascale", IRCCS, Via Mariano Semmola, 80131, Napoli, Italy.
  • Cavalcanti E; Laboratory Medicine Unit, Istituto Nazionale Tumori-IRCCS "Fondazione G. Pascale", 80131, Napoli, Italy.
  • Botti G; Scientific Direction, Istituto Nazionale Tumori-Irccs-Fondazione G. Pascale, 80131, Napoli, Italy.
  • Vaccari G; ISS-Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, 00161, Rome, Italy.
  • Portella G; Dipartimento Scienze Mediche Traslazionali, Università di Napoli "Federico II", 80131, Napoli, Italy.
  • Normanno N; Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori "Fondazione Giovanni Pascale", IRCCS, Via Mariano Semmola, 80131, Napoli, Italy. n.normanno@istitutotumori.na.it.
J Transl Med ; 19(1): 246, 2021 06 05.
Article in English | MEDLINE | ID: covidwho-1259200
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Prognostic study Topics: Vaccines / Variants Limits: Humans Country/Region as subject: Europa Language: English Journal: J Transl Med Year: 2021 Document Type: Article Affiliation country: S12967-021-02912-4

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Prognostic study Topics: Vaccines / Variants Limits: Humans Country/Region as subject: Europa Language: English Journal: J Transl Med Year: 2021 Document Type: Article Affiliation country: S12967-021-02912-4