Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.
Article activity feed
-
-
-
SciScore for 10.1101/2020.08.04.236893: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Trimmed alignments were converted to pileup format using samtools mpileup (v1.9)38, with anomalous read pairs retained (--count-orphans), base alignment quality disabled (--no-BAQ) and all bases considered, regardless of PHRED quality (--min-BQ 0). samtoolssuggested: (SAMTOOLS, RRID:SCR_002105)Up to twelve samples were multiplexed on a FLO-FLG001, FLO-MIN106D or FLO-PRO002 or flow-cell and sequenced on a GridION X5 or PromethION P24 device, respectively. PromethIONsuggested: …SciScore for 10.1101/2020.08.04.236893: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Trimmed alignments were converted to pileup format using samtools mpileup (v1.9)38, with anomalous read pairs retained (--count-orphans), base alignment quality disabled (--no-BAQ) and all bases considered, regardless of PHRED quality (--min-BQ 0). samtoolssuggested: (SAMTOOLS, RRID:SCR_002105)Up to twelve samples were multiplexed on a FLO-FLG001, FLO-MIN106D or FLO-PRO002 or flow-cell and sequenced on a GridION X5 or PromethION P24 device, respectively. PromethIONsuggested: (PromethION, RRID:SCR_017987)The RAMPART software package39 was used to monitor sequencing performance in real-time, with runs proceeding until a minimum ~200-fold coverage was achieved across all amplicons. RAMPARTsuggested: (Rampart, RRID:SCR_016742)Variant candidates identified by Illumina/ONT could then be considered concordant based on matching genome position, reference base and alternative base/s. Illumina/ONTsuggested: NoneResults from OddPub: Thank you for sharing your code and data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-