Multiple approaches for massively parallel sequencing of HCoV-19 (SARS-CoV-2) genomes directly from clinical samples

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   ScreenIT

   Jun 5, 2020

   SciScore for 10.1101/2020.03.16.993584: (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 Institutional Review Boards (IRB) of the First Affiliated Hospital of Guangzhou Medical University approved the clinical studies.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Sequencing was attempted on all samples regardless of Ct value including negative controls prepared from nuclease-free water and NA12878 human gDNA.	NA12878 suggested: Coriell Cat# GM12878, RRID:CVCL_7526)
   Software and Algorithms
   Sentences	Resources
   DNBs-based libraries were constructed and sequenced …

   SciScore for 10.1101/2020.03.16.993584: (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 Institutional Review Boards (IRB) of the First Affiliated Hospital of Guangzhou Medical University approved the clinical studies.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Sequencing was attempted on all samples regardless of Ct value including negative controls prepared from nuclease-free water and NA12878 human gDNA.	NA12878 suggested: Coriell Cat# GM12878, RRID:CVCL_7526)
   Software and Algorithms
   Sentences	Resources
   DNBs-based libraries were constructed and sequenced on the MGISEQ-2000 platform with paired-end 100 nt strategy using the same protocol described above, generating 37 Gb sequencing data for each sample on average.	MGISEQ-2000 suggested: (DNBSEQ-G400, RRID:SCR_017980)
   Identification of HCoV-19-like reads from Massively Parallel Sequencing data: For metatranscriptomic and hybrid capture sequencing data, total reads were first processed by Kraken v0.10.526 (default parameters) with a self-build database of Coronaviridae genomes (including SARS, MERS and HCoV-19 genome sequences downloaded from GISAID, NCBI and CNGB) to efficiently identify candidate viral reads with a loose manner.	Kraken suggested: (Kraken, RRID:SCR_005484)
   These candidate reads were further qualified with fastp v0.19.527 (parameters: -q 20 -u 20 - n 1 -l 50) and SOAPnuke v1.5.628 (parameters: -l 20 -q 0.2 -E 50 -n 0.02 -5 0 -Q 2 -G -d) to remove low-quality reads, duplications and adaptor contaminations.	SOAPnuke suggested: (SOAPnuke, RRID:SCR_015025)
   Low-complexity reads were next filtered by PRINSEQ v0.20.429 (parameters: -lc_method dust -lc_threshold 7).	PRINSEQ suggested: (PRINSEQ, RRID:SCR_005454)
   Assembling viral genome: HCoV-19-like reads of metatranscriptomic and hybrid capture sequencing data were de novo assembled with SPAdes (v3.14.0)31 using the default settings to obtain virus genome sequences.	SPAdes suggested: (SPAdes, RRID:SCR_000131)
   Due to the uneven read coverage in amplicon sequencing of HCoV-19, virus consensus sequences of amplicon samples were generated by Pilon v1.2332 (parameters: --changes - vcf --changes --vcf --mindepth 1 --fix all, amb).	Pilon suggested: (Pilon , RRID:SCR_014731)
   Assessment the coverage depth across the viral genome: HCoV-19-like reads of metatranscriptomic and hybrid capture sequencing data were aligned to the HCoV-19 reference genome (GISAID accession: EPI_ISL_402119) with BWA aln (v0.7.16)33.	BWA suggested: (BWA, RRID:SCR_010910)
   For each sample, we calculated the depth of coverage at each nucleotide position of the HCoV-19 reference genome with Samtools (v1.9)34 and scaled the values to the mean depth.	Samtools suggested: (SAMTOOLS, RRID:SCR_002105)
   Consistency in variants calling performance among methods: Except for amplicon sequencing samples, variants calling in metatranscriptomic and hybrid capture sequencing samples was performed in the previous BAM files of identified HCoV-19 reads after removing duplications from alignment output by Picard Markduplicates (http://broadinstitute.github.io/picard).	Picard suggested: (Picard, RRID:SCR_006525)
   After QC, we mapped high-quality reads to hg19 and removed human ribosomal RNA (rRNA) reads by SOAP2 v2.2142 (parameters: - m 0 -x 1000 -s 28 -l 32 -v 5 -r 1), and the remaining RNA reads were then aligned to hg19 by HISAT243 with default settings to identify non-rRNA human transcripts as previously described.	SOAP2 suggested: None
   Bracken45 (Bayesian Reestimation of Abundance with Kraken) was further applied to estimate microbial relative abundances based on taxonomic ranks of reads assigned by Kraken2.	Kraken2 suggested: None

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
   • 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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   Version published to 10.1101/2020.03.16.993584 on bioRxiv

   Mar 17, 2020