Abstract
IntroductionThe ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. An update to the V3 primer set was released on 18th June 2021 to address amplicon drop-off observed among the Delta variant of concern. Here, we report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs.MethodsWe utilized a matched set of 43 clinical samples and serially diluted positive controls that were amplified by ARTIC V3, V4 and optimized V4 primers and sequenced using GridION from the Oxford Nanopore Technologies'.ResultsWe observed a 0.5% to 46% increase in genome recovery in 67% of the samples when using the original V4 pooling strategy compared to the V3 primers. Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. When using the optimized protocol, we observed a 60% improvement in genome recovery across all samples and an increase in the average depth in amplicon 23 and 90. Consequently, ≥95% of the genome was recovered in 72% (n = 31) of the samples. However, only 60–70% of the genomes could be recovered in samples that had <28% genome coverage with the ARTIC V3 primers. There was no statistically significant (p > 0.05) correlation between Ct value and genome recovery.ConclusionUtilizing the ARTIC V4 primers, while increasing the primer concentrations for amplicons with drop-offs or low average read-depth, greatly improves genome recovery of Alpha, Beta, Delta, Eta and non-VOC/non-VOI SARS-CoV-2 variants.
Highlights
The ARTIC Network’s primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol
The most widely adopted targeted amplicon approach for SARS-CoV-2 genomic sequencing is the ARTIC protocol. This protocol was developed based on an earlier strategy for sequencing single-stranded Ribonucleic acid (RNA) viruses from high cycle threshold (Ct) clinical samples [14]
The V3 primer set contained additional alternate primers added to the V1 primer sets and provided over 50X coverage in all amplicons compared to V1 and V2 primer-sets [13]
Summary
The ARTIC Network’s primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. We report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs. The most widely adopted targeted amplicon approach for SARS-CoV-2 genomic sequencing is the ARTIC protocol This protocol was developed based on an earlier strategy for sequencing single-stranded RNA viruses from high cycle threshold (Ct) clinical samples [14]. It employed an early draft version of the SARS-CoV-2 genome and incorporated two sets of primer pools for efficient multiplexing [15, 16]. The reagents’ cost of SARS-CoV-2 WGS using Nanopore devices has been estimated to be between $11.50 to $35.88 for one sample when calculated based on 96 samples per sequencing run [18,19,20]
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