Abstract
A group of biologists, ARTIC Network, has proposed a multiplexed PCR primer set for whole-genome analysis of the novel coronavirus, SARS-CoV-2, soon after the start of COVID-19 epidemics was realized. The primer set was adapted by many researchers worldwide and has already contributed to the high-quality and prompt genome epidemiology of this rapidly spreading viral disease. We have also seen the great performance of their primer set and protocol; the primer set amplifies all desired 98 PCR amplicons with fairly small amplification bias from clinical samples with relatively high viral load. However, we also observed an acute drop of reads derived from some amplicons especially amplicon 18 and 76 in “pool 2” as a sample’s viral load decreases. We suspected this low coverage issue was due to dimer formation between primers targeting those two amplicons. Indeed, replacement of just one of those primers, nCoV-2019_76_RIGHT, to a newly designed primer resulted in a drastic improvement of coverages at both regions targeted by the amplicons 18 and 76. Given this result, we further replaced four primers in “pool 1” with each respective alternative. These modifications also improved coverage in eight amplicons particularly in samples with low viral load. The results of our experiments clearly indicate that primer dimer formation is one critical cause of coverage bias in ARTIC protocol. Importantly, some of the problematic primers are detectable by observing primer dimers in raw NGS sequence reads and replacing them with alternatives as shown in this study. We expect a continuous improvement of the ARTIC primer set will extend the limit for completion of SARS-CoV-2 genomes to samples with lower viral load, that supports better genomic epidemiology and mitigation of spread of this pathogen.
Footnotes
There were several errors about the genomic coorditates of primers in older versions.
