Abstract
BackgroundSince the first public genome of SARS-CoV-2, over 170,000 genome sequences of the virus have been shared by researchers world-wide (till November 1st 2020). Multiplex PCR targeting SARS-CoV-2 followed by massively parallel sequencing (MPS) and/or nanopore sequencing is a widely used strategy to recover the genome from primary samples. However, the bias of amplification among different amplicons should not be ignored, which might lead to uneven sequencing coverage on the viral genome.MethodsWe aim to develop a novel multiplex PCR panel to achieve an improved coverage evenness of SARS-CoV-2. We adapt long amplicons (~1000-bp) for the panel and thus reduced the number of primer pairs. The panel was validated with clinical samples and sequenced via MPS sequencing systems and a portable nanopore sequencing device MinION. We evaluated the full-genome coverage evenness and its dependence on viral loads of the long amplicon panel; we then compared it with a 98-plex panel provided by the ARTIC network. The accuracy to identify viral genomic variations based on the panel and sequencing with MinION was assessed.ResultsWe developed a two-pool 36-plex panel for full-genome sequencing of SARS-CoV-2, whose amplicon size ranged from 880 to 1027 bp. For samples with a <30 Ct value, >90% viral genome could be recovered with a high sequencing depth (>0.2 mean depth) by using the long-amplicon panel (n = 36), compared with 79-88% highly covered genome region for the ARTIC panel (n = 5). The coverage evenness of the long-amplicon panel was also less affected by low viral titers and not dependent on sequencing data amount. With MinION sequencing, the consensus viral genomes could be reliably recovered. However, a high false positive rate was observed to identify sub-clonal genomic variations with a <0.6 frequency.ConclusionA novel multiplex PCR panel for full-genome sequencing of SARS-CoV-2 with improved coverage evenness and low requirement of data throughput was validated with clinical samples. Amplification of SARS-CoV-2 with the panel followed by MinION sequencing could generate reliable consensus genome sequences, but the detection of non-dominating viral populations within host is error-prone.
Complete Genome Sequences of Three African Foot-and-Mouth Disease Viruses from Clinical Samples Isolated in 2009 and 2010