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 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: COVID-19; SARS-CoV-2; genomic surveillance; next generation sequencing; whole-genome sequencing.

Figure 1.

Plots of coverage according to evaluated methods and Cycle threshold (Ct) values groups. Dotted lines indicate the minimum depth of 10× for Illumina methods and 20× for ONT method. Missing sites in the genome are those with a coverage <10× for Illumina methods and <20× for the ARTIC-ONT method. Using an R script, these plots were constructed via ggplot2 from depth files generated by BEDtools from output aligned bam files of each specific-method pipeline.

Figure 2.

Mismatch count between consensus sequences generated by each method compared two by two for each sample. These matrices were generated only from consensus with determined bases for more than 99 per cent of the genome. If one sequence of the two had more than 1 per cent of undetermined bases (N), comparison was not assessed, grey tiles. Blue tiles correspond to perfect identity and orange tile correspond to mismatches, the number of mismatches is indicated inside the tile. Matrices were generated with an R script using Decipher (alignment), ape (distance matrices), and ggplot2 (charts) libraries. Of note, undetermined bases and deletions were not considered in the calculation of mismatches. * For the sample #19: the position 533 is undetermined by ARTIC method, and therefore no SNP is observed between ARTIC and CleanPlex methods.