Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation

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 commercialized kits including a hybridization 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, with mNGS and RVOP producing the most complete genomes. For mid viral loads, 2/8 and 1/8 genomes were incomplete (<99%) with mNGS and both CleanPlex and RVOP, respectively. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques yielding complete genomes for 7/8 samples. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in two 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 34-nt deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a default 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 regular updates of the technique according to SARS-CoV-2 evolution.

Characterization of SARS-CoV-2 ORF6 deletion variants detected in a nosocomial cluster during routine genomic surveillance, Lyon, France

“…After DNAse treatment (Life Technologies, Carlsbad, CA, USA), samples underwent random amplification using Whole Transcriptome Amplification (WTA2 kit, Sigma-Aldrich, Darmstadt, DE) before sequencing on an Illumina NextSeqTM 550 with mid-output 2x150 flow cell. Importantly, the variants displaying an ORF6 deletion were confirmed by 3 other techniques, including capture-and amplicon-based strategies [23].…”

Role of Multiomics Data to Understand Host–Pathogen Interactions in COVID-19 Pathogenesis

“… 54 Moreover, the metagenomic NGS (mNGS) protocol that allows detection of SARS-CoV-2 and other associated pathogens present in a clinical sample at once has aided the discovery of coinfections that would have been otherwise missed. 55 In a similar study, a higher abundance of Propionibacteriaceae and reductions in Corynebacterium accolens was observed in COVID-19 negative patients compared to COVID-19 positive patients. 56 Next-gen immunosequencing performed from blood collected from COVID-19 positive patients as well as recovered patients has been used to generate a library of more than 14 billion B and T-cell receptor sequences to decode host humoral responses generated postinfection.…”

“…Another recently developed in-house metagenomic NGS (mNGS) can detect SARS Cov2 and associated all other pathogens in a clinical sample, which eliminates the necessity of using multiple test kits to detect comorbidity. 55 …”

Target Capture Sequencing of SARS-CoV-2 Genomes Using the ONETest Coronaviruses Plus

“…Alternatively, SARS-CoV-2 genome sequencing can be accomplished using probe-based liquid-phase hybridization followed by NGS 3,7,8 . A major appeal of target capture NGS methodologies is its capacity to enrich samples for a practically limitless repertoire of genetic loci without needing to constantly update the primers and or deal with multiplexing issues encountered with amplicon-based approaches.…”