Disentangling primer interactions improves SARS-CoV-2 genome sequencing by multiplex tiling PCR

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.

Oligonucleotide Capture Sequencing of the SARS-CoV-2 Genome and Subgenomic Fragments from COVID-19 Individuals

“…ARTIC V1 primer set, worked well for full-length genome recovery with relatively high viral load (Ct < 25) in clinical qPCR tests, as certain primer pairs were under performing. The updated V3 and NIID-1 primer sets addressed this problem and were shown to work well with Ct values in clinical qPCR from 25 to 30 [4]. A multiplex amplicon-based approaches by CDC 23 where the effectively generating full length genome sequences <Ct of 33 although Ct. values between 30 and 33, genome recovery varied between samples.…”

Next generation sequencing of SARS-CoV-2 genomes: challenges, applications and opportunities

“…While amplicon sequencing is theoretically convenient and cheap, it presents some limitations which should be considered. Firstly, because of differences in primer efficiency, or possible variants in the primer annealing regions, amplification across the genome can be biased, with decreased coverage in specific genomic regions (see V1 version of ARTIC protocol [ 88 , 89 ]) and/or 3′ and 5′ UTRs regions missed altogether (see Supplementary Table S3 available online at https://academic.oup.com/bib ) leading to an incomplete assembly. Moreover, since the primers are designed on the reference SARS-CoV-2 genome sequence, this approach may not identify large structural variants and can present systematic limitations in the presence of high levels of genomic divergence.…”

Sensitive Recovery of Complete SARS-CoV-2 Genomes from Clinical Samples by Use of Swift Biosciences’ SARS-CoV-2 Multiplex Amplicon Sequencing Panel

“…Whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a better understanding of the virus’s origin, transmission, and evolution ( 1 – 6 ). Multiplex amplicon sequencing for viral WGS is a preferred approach to library preparation since it is simple, sensitive, cost-effective, and scalable ( 7 , 8 ). However, balancing multiplexed primers to achieve high sensitivity and even coverage can be difficult ( 8 ), and superlative analytical sensitivity is not assured.…”

“…Multiplex amplicon sequencing for viral WGS is a preferred approach to library preparation since it is simple, sensitive, cost-effective, and scalable ( 7 , 8 ). However, balancing multiplexed primers to achieve high sensitivity and even coverage can be difficult ( 8 ), and superlative analytical sensitivity is not assured. Here, we evaluated the Swift Biosciences’ single-tube SARS-CoV-2 multiplex amplicon sequencing panel for the recovery of genomes from low-viral-load samples (threshold cycle [ C T ] > 26 on a Hologic Panther Fusion system).…”