Abstract
Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample’s viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network’s original (V1) and modified (V3) primer set.
Highlights
The realtime surveillance of pathogen genome sequences during an outbreak enables monitoring of numerous epidemical factors such as pathogen adaptation and transmission chains in local to even global scale [1, 2]. Since it was first identified in Hubei, China in December 2019 [3], the novel coronavirus, SARS-CoV-2, responsible for the atypical respiratory illness COVID-19, has become a major concern for the medical community around the world
The results indicated that preventing primer dimerformation is an effective measure to improve coverage bias in the ARTIC Network’s SARS-CoV2 genome sequencing protocol, and may be applicable to other PrimalSeq methods in general
We identified an additional 13 primer interactions using in silico analysis (Fig 2A and 2B)
Summary
The realtime surveillance of pathogen genome sequences during an outbreak enables monitoring of numerous epidemical factors such as pathogen adaptation and transmission chains in local to even global scale [1, 2]. In January 2020, a group of biologists comprising the ARTIC Network (https://artic.network/), designed 196 primers (98 pairs) (https://github.com/articnetwork/artic-ncov2019/tree/master/primer_schemes/nCoV-2019/V1) for targeted amplification of the SARS-CoV-2 genome by multiplexing PCR. The ARTIC primer set V1 and the published protocol [7] worked well for samples with a relatively high viral load (Ct < 25 in clinical qPCR tests). For these samples, all designated amplicons are amplified with an acceptable level of coverage bias for subsequent NGS analysis. The results indicated that preventing primer dimerformation is an effective measure to improve coverage bias in the ARTIC Network’s SARS-CoV2 genome sequencing protocol, and may be applicable to other PrimalSeq methods in general
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