tetano
Editor, Senior Moderator
J Transl Med
. 2021 Jun 5;19(1):246.
doi: 10.1186/s12967-021-02912-4.
SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system
Anna Maria Rachiglio[SUP] #[/SUP][SUP] 1 [/SUP], Luca De Sabato[SUP] #[/SUP][SUP] 2 [/SUP], Cristin Roma[SUP] 1 [/SUP], Michele Cennamo[SUP] 3 [/SUP], Mariano Fiorenza[SUP] 3 [/SUP], Daniela Terracciano[SUP] 3 [/SUP], Raffaella Pasquale[SUP] 1 [/SUP], Francesca Bergantino[SUP] 1 [/SUP], Ernesta Cavalcanti[SUP] 4 [/SUP], Gerardo Botti[SUP] 5 [/SUP], Gabriele Vaccari[SUP] 2 [/SUP], Giuseppe Portella[SUP] 3 [/SUP], Nicola Normanno[SUP] 6 [/SUP]
Affiliations
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.
Keywords: Campania region; Covid-19; Next generation sequencing; SARS-CoV-2 genome.
. 2021 Jun 5;19(1):246.
doi: 10.1186/s12967-021-02912-4.
SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system
Anna Maria Rachiglio[SUP] #[/SUP][SUP] 1 [/SUP], Luca De Sabato[SUP] #[/SUP][SUP] 2 [/SUP], Cristin Roma[SUP] 1 [/SUP], Michele Cennamo[SUP] 3 [/SUP], Mariano Fiorenza[SUP] 3 [/SUP], Daniela Terracciano[SUP] 3 [/SUP], Raffaella Pasquale[SUP] 1 [/SUP], Francesca Bergantino[SUP] 1 [/SUP], Ernesta Cavalcanti[SUP] 4 [/SUP], Gerardo Botti[SUP] 5 [/SUP], Gabriele Vaccari[SUP] 2 [/SUP], Giuseppe Portella[SUP] 3 [/SUP], Nicola Normanno[SUP] 6 [/SUP]
Affiliations
- PMID: 34090468
- DOI: 10.1186/s12967-021-02912-4
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.
Keywords: Campania region; Covid-19; Next generation sequencing; SARS-CoV-2 genome.