Your browser doesn't support javascript.
Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation.
Charre, Caroline; Ginevra, Christophe; Sabatier, Marina; Regue, Hadrien; Destras, Grégory; Brun, Solenne; Burfin, Gwendolyne; Scholtes, Caroline; Morfin, Florence; Valette, Martine; Lina, Bruno; Bal, Antonin; Josset, Laurence.
  • Charre C; Laboratoire de Virologie, Institut des Agents Infectieux (IAI), Hospices Civils de Lyon, Groupement Hospitalier Nord, Lyon cedex 4, France.
  • Ginevra C; Université de Lyon, Université Claude Bernard Lyon 1 (UCBL1), Lyon, France.
  • Sabatier M; Centre de recherche en cancérologie (CRCL), INSERM U1052- 69008, Lyon, France.
  • Regue H; CIRI, Centre International de Recherche en Infectiologie, (Team pathogenesis of Legionella), Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon 69007, France.
  • Destras G; Hospices Civils de Lyon, Centre National de Référence des Légionelles, Lyon, France.
  • Brun S; Laboratoire de Virologie, Institut des Agents Infectieux (IAI), Hospices Civils de Lyon, Groupement Hospitalier Nord, Lyon cedex 4, France.
  • Burfin G; Université de Lyon, Université Claude Bernard Lyon 1 (UCBL1), Lyon, France.
  • Scholtes C; Université de Lyon, Virpath, CIRI, INSERM U1111, CNRS UMR5308, ENS Lyon, Université Claude Bernard Lyon 1, Lyon, France.
  • Morfin F; Laboratoire de Virologie, Institut des Agents Infectieux (IAI), Hospices Civils de Lyon, Groupement Hospitalier Nord, Lyon cedex 4, France.
  • Valette M; Université de Lyon, Université Claude Bernard Lyon 1 (UCBL1), Lyon, France.
  • Lina B; Laboratoire de Virologie, Institut des Agents Infectieux (IAI), Hospices Civils de Lyon, Groupement Hospitalier Nord, Lyon cedex 4, France.
  • Bal A; Université de Lyon, Université Claude Bernard Lyon 1 (UCBL1), Lyon, France.
  • Josset L; Université de Lyon, Virpath, CIRI, INSERM U1111, CNRS UMR5308, ENS Lyon, Université Claude Bernard Lyon 1, Lyon, France.
Virus Evol ; 6(2): veaa075, 2020 Jul.
Article in English | MEDLINE | ID: covidwho-814175
ABSTRACT
Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next-Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty-four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing an in-house metagenomic NGS (mNGS) protocol and two newly commercialised kits including a hybridisation capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP), and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples (n = 8), with mNGS and RVOP producing the most complete genomes. For mid viral loads (Ct 20-25), amplicon-based enrichment methods led to genome coverage >99 per cent for all samples while 1/8 sample sequenced with RVOP and 2/8 samples sequenced with mNGS had a genome coverage below 99 per cent. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in three samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34nt-deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a loss of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer-annealing or probe-hybridizing regions and would require further validation using unbiased metagenomic sequencing.
Keywords

Full text: Available Collection: International databases Database: MEDLINE Type of study: Experimental Studies / Prognostic study Language: English Journal: Virus Evol Year: 2020 Document Type: Article Affiliation country: Ve

Similar

MEDLINE

...
LILACS

LIS


Full text: Available Collection: International databases Database: MEDLINE Type of study: Experimental Studies / Prognostic study Language: English Journal: Virus Evol Year: 2020 Document Type: Article Affiliation country: Ve