Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols

Abstract

Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with PCR-tiling approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, including human and bacterial contamination, as well as the presence of reads derived from the viral sub-genomic RNAs.

High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination

“…Thus, the long amplicon protocol appears to be effective in detecting spike variants. Our observation was also supported by a recent publication [ 39 ]. Brejova et al compared the sequencing results obtained from the libraries containing SARS-CoV-2 samples made of 400 bp (primer set I), 2000 bp, and 2500 bp (primer set III) amplicon pools to conclude that sequencing long amplicons clearly outperforms shorter amplicons in terms of lower coverage variation and overall quality of the consensus sequences.…”

Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs

“…Oxford Nanopore protocols SARS-CoV2 PCR tailing based are suggested to sequence the coronavirus genome directly from nasopharyngeal or oropharyngeal swabs. In order to cover the whole genome, these protocols, exploiting the technology based on an array of nanopores, use a multiplex primers scheme , which generate longer amplicons and tend to produce close-to-finished genomes more quickly than short reads based sequencing platforms [9]. Moreover, despite a single nanopore long read can have a more relatively higher error rate than short read, studies that compare the genome assembly derived from both technologies demonstrated that through Nanopore it is possible to achieve highly accurate consensus single nucleotide variant (SNV) calling with >99% sensitivity and >99% precision with a minimum of about 60-fold coverage [9].…”

“…On other hand, these protocols produce a higher percentage of reads that are not analyzable by the bioinformatic pipelines, likely due to a combination of fragmentation of synthesized molecules and prematurely aborted molecules during sequencing [9]. Brejová et coworkers dissected the genome assembly generated by sequencing of amplicons derived from samples of the same biological material using schemes of primers set, which produced long reads of 400-bp, 2-kb, and 2.5 kb.…”

“…It has been developed by the Artic Network for the sequencing of Ebola, Zika, and Chikungunya genomes [7,8]. Consequently, it was promptly adjusted for rapid sequence determination of SARS-CoV-2 RNA, in January 2020 [9]. Additional studies have led to improvements of this protocol, such as the simplification of the sequencing library preparation and increased sample multiplexing (up to 96), which has brought down the costs of the sequencing for a single sample to about GBP 10 [9].…”

VirPool: Model-Based Estimation of SARS-CoV-2 Variant Proportions in Wastewater Samples

“…The samples originated from oropharyngeal swabs collected in January 2021. The sample processing and sequencing library construction were carried out as described earlier (Brejová et al, 2021), generally following the COVID-19 virus protocol (PTC 9096 v109 revF 06Feb2020; Oxford Nanopore Technologies, Oxford, UK) with some modifications. After RNA extraction for each sample, SARS-CoV-2 RNA was quantified by an RT-qPCR assay carried on QuantStudio™ 5 Real-Time PCR System (Applied Biosystem, Foster City, California, USA).…”