Whole‐genome sequencing of SARS‐CoV‐2: Comparison of target capture and amplicon single molecule real‐time sequencing protocols

Abstract

Fast, accurate sequencing methods are needed to identify new variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) genome. Single‐molecule real‐time (SMRT) Pacific Biosciences (PacBio) provides long, highly accurate sequences by circular consensus reads. This study compares the performance of a target capture SMRT PacBio protocol for whole‐genome sequencing (WGS) of SARS‐CoV‐2 to that of an amplicon PacBio SMRT sequencing protocol. The median genome coverage was higher ( p < 0.05) with the target capture protocol (99.3% [interquartile range, IQR: 96.3–99.5]) than with the amplicon protocol (99.3% [IQR: 69.9–99.3]). The clades of 65 samples determined with both protocols were 100% concordant. After adjusting for C t values, S gene coverage was higher with the target capture protocol than with the amplicon protocol. After stratification on C t values, higher S gene coverage with the target capture protocol was observed only for samples with C t > 17 ( p < 0.01). PacBio SMRT sequencing protocols appear to be suitable for WGS, genotyping, and detecting mutations of SARS‐CoV‐2.

Whole‐genome single molecule real‐time sequencing of SARS‐CoV‐2 Omicron

“…The proportion of sample sequence failures obtained with the target capture method in this study of Omicron variants was similar to that obtained for the Alpha/Delta variants 27 and the complete genome coverages of the Omicron (median >98%) and Alpha/Delta variants (median >99%) were also similar 27 . However, the proportion of samples with a complete genome coverage of >95% was slightly lower (55%) than for the Alpha/Delta variants (63%), perhaps because the Omicron samples of this study had lower viral loads.…”

“…We used the PacBio HiFiViral for SARS‐CoV‐2 workflow, High‐Throughput Multiplexing 1.2 kb Amplicons for Full‐Viral Genome Sequencing of SARS‐CoV‐2, based on 29 1.2 kb amplicons across the SARS‐CoV‐2 genome (hereafter referred to as amplicon) with improved primers for amplicons A1, A5, A10, A15, A21, A24, and A28, which have mutation‐induced reduced coverage in Omicron variants (Table 1). The protocol was done as previously described 27 . Briefly, we first generated cDNAs and then obtained the 29 amplicons by two multiplex PCRs (with two primers pools, Pool 1 and Pool 2).…”

“…We used the SARS‐CoV‐2 Enrichment (PacBio) kit following the PacBio HiFiViral for SARS‐CoV‐2 workflow, High‐Throughput Multiplexing for Full‐Viral Genome Sequencing of SARS‐CoV‐2 version 3 (hereafter referred to as target capture). This protocol uses molecular inversion probes with a 675 bp target insert and was conducted as previously described 27 . Briefly, the first step was simultaneous complementary DNA (cDNA) synthesis and probe hybridization, followed by circularization and barcoded amplification.…”

“…Next generation sequencing protocols, mostly based on multiplex amplicon or hybrid capture methods running on the Illumina and Oxford Nanopore Technologies (ONT) platforms, 17‐23 have been developed to study the genomic diversity of SARS‐CoV‐2 worldwide 24‐26 . We have recently shown that the Pacific Biosciences (PacBio) single molecule real‐time (SMRT) sequencing method, which provides accurate whole genome sequences (WGS), can be used for genotyping and detecting mutations in Alpha and Delta variants 27 . However, no Omicron variants were included in this study.…”

Hybrid-Capture Target Enrichment in Human Pathogens: Identification, Evolution, Biosurveillance, and Genomic Epidemiology

“…Expectedly, they found that taxon-specific panels produced the best results compared to broader-range panels (e.g., respiratory viruses panel). Beyond the choice of panels, studies also investigated and verified the efficacy of HC for SARS-CoV-2 sequences across bioinformatic pipelines [ 241 ], sequencing platforms [ 242 , 243 ], and even library preparation methods [ 244 ]. Generally, molecular techniques (e.g., metagenomic sequencing, HC, and PCR) performed comparably, but in SARS-CoV-2, as the pathogen has a relatively small genome (~30 kb), enrichment of sequences can be executed using dedicated primers for the entire genome (e.g., ARCTIC 4.1).…”