Overall results of sequencing of SARS-CoV-2 using an amplicon-based method and a capture-based method for enrichment.

a) Violin plot and boxplot of the median read depth divided by cohorts. (For Amplicon enrichment, Wilcoxon test; p p = 0.92). b,c) Genome coverage, percentage of the genome with a read depth over 10. Each data point corresponds to one sample. Amplicon: ARTIC panel, amplicon-based enrichment. Capture: KAPA RNA HyperCap, capture-based enrichment.</p

S1 Dataset -

To control the SARS-CoV-2 pandemic, healthcare systems have focused on ramping up their capacity for epidemiological surveillance through viral whole genome sequencing. In this paper, we tested the performance of two protocols of SARS-CoV-2 nucleic acid enrichment, an amplicon enrichment using different versions of the ARTIC primer panel and a hybrid-capture method using KAPA RNA Hypercap. We focused on the challenge of the Omicron variant sequencing, the advantages of automated library preparation and the influence of the bioinformatic analysis in the final consensus sequence. All 94 samples were sequenced using Illumina iSeq 100 and analysed with two bioinformatic pipelines: a custom-made pipeline and an Illumina-owned pipeline. We were unsuccessful in sequencing six samples using the capture enrichment due to low reads. On the other hand, amplicon dropout and mispriming caused the loss of mutation G21987A and the erroneous addition of mutation T15521A respectively using amplicon enrichment. Overall, we found high sequence agreement regardless of method of enrichment, bioinformatic pipeline or the use of automation for library preparation in eight different SARS-CoV-2 variants. Automation and the use of a simple app for bioinformatic analysis can simplify the genotyping process, making it available for more diagnostic facilities and increasing global vigilance.</div

Histograms of discrepancies in final consensus sequences.

a) Base mismatches in all studied samples using the amplicon-enrichment method or capture-enrichment method. b-c) Discrepancies using two different bioinformatic pipelines, a customised bioinformatic pipeline and Illumina® DRAGEN™ COVID lineage app. b) For ARTIC panel, amplicon-based enrichment. c) For KAPA RNA HyperCap, capture-based enrichment.</p

Custom protocol for RT-PCR amplification of SARS-CoV-2 RNA.

Custom protocol for RT-PCR amplification of SARS-CoV-2 RNA.</p

Evaluation of the effectiveness of two methods of enrichment.

a-b) Genome coverage per variant. Delta samples in the amplicon enrichment showed the lowest genome coverage. c-d) Percentage of samples with less than 10 reads on each base. Areas with dips in coverage were identified, with a notable peak consistent of Delta samples at the beginning of the spike gene. Amplicon: ARTIC panel, amplicon-based enrichment. Capture: KAPA RNA HyperCap, capture-based enrichment.</p

Phylogenetic tree for all samples analysed in the study.

Samples are coloured by clade, with undesignated recombinant samples shown in yellow. The tip label indicates the Pango lineage. Tree generated by Neighbour-Joining method; Maximum Composite Likelihood. Amplicon: ARTIC panel, amplicon-based enrichment. Capture: KAPA RNA HyperCap, capture-based enrichment.</p

Allele frequency plot by nucleotide of SARS-CoV-2 genome.

a) ARTIC panel, amplicon-based enrichment. b) KAPA RNA HyperCap, capture-based enrichment. Each point represents an SNP, while each line represents a LOESS local regression for each sample. One colour per sample. c) Violin plot and boxplot of the number of SNPs per sample at low frequency (0.2–0.9) or high frequency (>0.9).</p

ARTIC primers (v3 & v4.1) used for amplicon-based enrichment.

ARTIC primers (v3 & v4.1) used for amplicon-based enrichment.</p

Variant allele frequency plot for three possible undesignated recombinant samples.

Only BA.1-specific and BA.2-specific mutations were plotted, with the amplicon enrichment (ARTIC panel) on the left (ABE) and the capture enrichment (KAPA RNA HyperCap) on the right (CBE).The high allele frequency, with most SNPs called with over 95% allele frequency, suggesting recombination and not co-infection. Samples 2 and 3 (panels c-f) show likely the same breakpoint, suggesting both coming from the same origin. The lines represent a LOESS local regression for the allele frequency of each variant.</p

Coverage and depth per sample using all enrichment methods and bioinformatic analysis.

Coverage and depth per sample using all enrichment methods and bioinformatic analysis.</p