A benchmarking study of SARS-CoV-2 whole-genome sequencing protocols using COVID-19 patient samples
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SciScore for 10.1101/2020.11.10.375022: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics statement: The study was approved by the Institutional Review Board (IRB number 5200127) and the Institutional Biosafety Committee (IBC) of the Loma Linda University (LLU).
IACUC: Ethics statement: The study was approved by the Institutional Review Board (IRB number 5200127) and the Institutional Biosafety Committee (IBC) of the Loma Linda University (LLU).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 SARS-CoV-2 WGS library construction using QIAseq WGSsuggested: NoneQIAseqsuggested: NoneSARS-CoV-2 WGS library construction … SciScore for 10.1101/2020.11.10.375022: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics statement: The study was approved by the Institutional Review Board (IRB number 5200127) and the Institutional Biosafety Committee (IBC) of the Loma Linda University (LLU).
IACUC: Ethics statement: The study was approved by the Institutional Review Board (IRB number 5200127) and the Institutional Biosafety Committee (IBC) of the Loma Linda University (LLU).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 SARS-CoV-2 WGS library construction using QIAseq WGSsuggested: NoneQIAseqsuggested: NoneSARS-CoV-2 WGS library construction using Tecan Trio RNA-Seq library protocol (Protocol 4): Eight RNA samples isolated from fresh and frozen specimens were used for Tecan Trio RNA-seq library construction (NuGEN/Tecan), following the NuGEN protocol with integrated DNase treatment. SARS-CoV-2suggested: (Active Motif Cat# 91351, RRID:AB_2847848)SARS-CoV-2 WGS library construction using QIAseq WGSsuggested: NoneQIAseqsuggested: NoneA Kraken244 database was built based on the complete genomes in the NCBI RefSeq database for archaea, bacteria, protozoa, fungi, human and viruses (SARS-COV-2 genome included). RefSeqsuggested: (RefSeq, RRID:SCR_003496)To summarize the read mapping percentages to multiple taxa, the trimmed reads were classified into human, SARS-CoV-2, bacterial, and remaining reads (e.g., unclassified, archaeal, viral, fungi, protozoa) by using the Kraken2 database. Kraken2suggested: NoneSARS-Co-V-2 SNV variant calling and generation of consensus SNV variants: Variants were called on the bam files by VarScan 2 (v2.4.4) and BCFTools (v1.9). VarScansuggested: (VARSCAN, RRID:SCR_006849)To accurately identify SNVs, we used samtools mpileup (parameters: -A -d 20000 -Q 0) and varscan2 (v2.4.4) (parameters: --p-value 0.99 –variants). samtoolssuggested: (SAMTOOLS, RRID:SCR_002105)The variant fasta files generated from the same clinical sample but different protocols were piled up using Jalview (v2.11.1.0)47 alignment tool, from which one consensus fasta file was compiled for each clinical sample. Jalviewsuggested: (Jalview, RRID:SCR_006459)Results from OddPub: Thank you for sharing your code and data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Although the primer-panel based target amplicon sequencing has been shown as a cost-effective approach for sequencing the clinical COVID-19 samples to discover the individual genetic diversity29, we found there were some limitations for the ARTIC V3 amplicon-based target whole-amplification protocols. First, by design, the current ARTIC V3 amplicons only covered genome regions from positions 30 to 29836, which would make it impossible for the ARTIC V3 amplicon-based protocols to detect a SNV outside of the PCR amplified regions. This scenario actually occurred in our benchmarking study and we found that a consensus variant, g.29868 G>A in sample NP29, was consistently detected by protocols P2, P3, and P4, but was missed by P1 and P7 (Fig. 5a, c&d). Second, a single-base mismatch between the primer and template may produce a PCR error such as chimeric PCR amplification30, which might lead to a false SNV call. For example, we found that P7, at low viral input, called a unique “false” SNV (g.28321 G>T) with almost 100% allele frequency and >1,000X coverage (Suppl. Fig. 13). However, this putative SNV was not detected in the same clinical sample prepared using either P7 at high viral input (1M) or P1, P2, P3 and P4 (Fig. 5c, Suppl. Fig. 13) at any input. Third, PCR amplified primer-originated “contaminated” sequences associated with the Qiagen protocols P1 and P7 may lead to an error in SNV calling. Coincidently, we had a consensus SNV (g.6543 C>T) which was within the overlappin...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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.
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