1 - read pre-processing
Version 1

Preprocessing of raw SARS-CoV-2 reads

The raw reads available so far are generated from bronchoalveolar lavage fluid (BALF) and are metagenomic in nature: they contain human reads, reads from potential bacterial co-infections as well as true COVID-19 reads.

Live Resources

usegalaxy.org usegalaxy.eu usegalaxy.org.au usegalaxy.be
Galaxy workflow Galaxy workflow Galaxy workflow Galaxy workflow
Galaxy history Galaxy history Galaxy history Galaxy history

What's the point?

Assess quality of reads, remove adapters and remove reads mapping to human genome.

The outline

Illumina and Oxford nanopore reads are pulled from the NCBI SRA (links to SRA accessions are available here). They are then processed separately as described in the workflow section.

Inputs

> :boom: If you experience problems downloading data from NCBI SRA, use Galaxy history pre-populated with inputs as described in "Alternate Workflow" section below.

Only SRA accessions are required for this analysis. The described analysis was performed with all SRA SARS-CoV accessions available as of Feb 20, 2020:

  1. Illumina reads

SRR10903401 SRR10903402 SRR10971381

  1. Oxford Nanopore reads

SRR10948550 SRR10948474 SRR10902284

Outputs

This workflow produces three outputs that are used in two subsequent analyses:

# Output Used in
1. A combined set of adapter-free Illumina reads without human contamination Assembly
2. A combined set of Oxford Nanopore reads without human contamination Assembly
3. A collection of adapter-free Illumina reads from which human reads have not been removed Variation detection

The history and the workflow

A Galaxy workspace (history) containing the most current analysis can be imported from here.

The publicly accessible workflow can be downloaded and installed on any Galaxy instance. It contains version information for all tools used in this analysis.

The workflow performs the following steps:

Illumina

  • Illumina reads are QC'ed and adapter sequences are removed using fastp
  • Quality metrics are computed and visualized using fastqc and multiqc
  • Reads are mapped against human genome version hg38 using bwa mem
  • Reads that do not map to hg38 are filtered out using samtools view
  • Reads are converted back to fastq format using samtools fastx

Oxford nanopore

  • Reads are QC'ed using nanoplot
  • Quality metrics are computed and visualized using fastqc and multiqc
  • Reads are mapped against human genome version hg38 using minimap2
  • Reads that do not map to hg38 are filtered out using samtools view
  • Reads are converted back to fastq format using samtools fastx

BioConda

Tools used in this analysis are also available from BioConda:

Name Link
sra-tools Anaconda-Server Badge
fastqc Anaconda-Server Badge
multiqc Anaconda-Server Badge
fastp Anaconda-Server Badge
nanoplot Anaconda-Server Badge
bwa Anaconda-Server Badge
picard Anaconda-Server Badge
samtools Anaconda-Server Badge

Alternate Workflow

An alternate starting point has been created for those not wanting to wait for the data to be downloaded from the NCBI SRA. (This can especially be an issue in Australia or Europe.)

There is a shared history containing all of the starting data in appropriate collections and an alternate workflow able to make use of this alternate input. Apart from a slightly different starting point, the workflow and the outputs it produces are identical to that above.

usegalaxy.org usegalaxy.eu usegalaxy.org.au usegalaxy.be
Galaxy input history Galaxy input history Galaxy input history Galaxy input history
Galaxy alternate workflow Galaxy alternate workflow Galaxy alternate workflow Galaxy alternate workflow
Galaxy final history Galaxy final history Galaxy final history Galaxy history

Inputs

ID Name Description Type
bed_file bed_file runtime parameter for tool Filter SAM or BAM, output SAM or BAM n/a

Steps

ID Name Description
0 List of Illumina accessions
1 List of ONT accessions
2 Illumina data toolshed.g2.bx.psu.edu/repos/iuc/sra_tools/fasterq_dump/2.10.4
3 ONT data toolshed.g2.bx.psu.edu/repos/iuc/sra_tools/fasterq_dump/2.10.4
4 fastp: Trimmed Illumina Reads toolshed.g2.bx.psu.edu/repos/iuc/fastp/fastp/0.19.3.3
5 NanoPlot toolshed.g2.bx.psu.edu/repos/iuc/nanoplot/nanoplot/1.28.2+galaxy1
6 FastQC toolshed.g2.bx.psu.edu/repos/devteam/fastqc/fastqc/0.72
7 Map with minimap2 toolshed.g2.bx.psu.edu/repos/iuc/minimap2/minimap2/2.12
8 MultiQC toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.7
9 Map with BWA-MEM toolshed.g2.bx.psu.edu/repos/devteam/bwa/bwa_mem/0.7.17.1
10 MultiQC toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.7
11 Filter SAM or BAM, output SAM or BAM toolshed.g2.bx.psu.edu/repos/devteam/samtool_filter2/samtool_filter2/1.8
12 Filter SAM or BAM, output SAM or BAM toolshed.g2.bx.psu.edu/repos/devteam/samtool_filter2/samtool_filter2/1.8
13 MergeSamFiles toolshed.g2.bx.psu.edu/repos/devteam/picard/picard_MergeSamFiles/2.18.2.1
14 MergeSamFiles toolshed.g2.bx.psu.edu/repos/devteam/picard/picard_MergeSamFiles/2.18.2.1
15 ONT filtered reads toolshed.g2.bx.psu.edu/repos/iuc/samtools_fastx/samtools_fastx/1.9+galaxy1
16 Illumina filtered reads toolshed.g2.bx.psu.edu/repos/iuc/samtools_fastx/samtools_fastx/1.9+galaxy1

Outputs

ID Name Description Type
list_paired list_paired n/a input
output_collection output_collection n/a input
output_collection_other output_collection_other n/a input
log log n/a txt
list_paired list_paired n/a input
output_collection output_collection n/a input
output_collection_other output_collection_other n/a input
log log n/a txt
output_paired_coll output_paired_coll n/a input
report_html report_html n/a html
report_json report_json n/a json
output_html output_html n/a html
nanostats nanostats n/a txt
nanostats_post_filtering nanostats_post_filtering n/a txt
read_length read_length n/a png
log_read_length log_read_length n/a png
html_file html_file n/a html
text_file text_file n/a txt
alignment_output alignment_output n/a bam
stats stats n/a input
html_report html_report n/a html
bam_output bam_output n/a bam
stats stats n/a input
html_report html_report n/a html
output1 output1 n/a sam
output1 output1 n/a sam
outFile outFile n/a bam
outFile outFile n/a bam
nonspecific nonspecific n/a fasta
forward forward n/a fasta
reverse reverse n/a fasta
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Created: 17th Sep 2020 at 10:38

Last used: 22nd Oct 2020 at 03:45

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