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Aims:

• Implement an end-to-end bioinformatics workflow that is reproducible, robust, scalable and compute infrastructure agnostic

• Leverage from the host plant antiviral response pathway to increase sensitivity and specificity of pathogen detections

• Prevent or minimise the reporting of cross-sample contaminations owing to index hopping events (false positive detections)

Pre-requisites

• Installed conda3 or miniconda3 ( https://docs.conda.io/projects/conda/en/latest/user-guide/install/linux.html )

• Basic unix command line knowledge (example: https://researchcomputing.princeton.edu/education/external-online-resources/linux ; https://swcarpentry.github.io/shell-novice/ )

• Familiarity with one unix text editors (example Vi/Vim or Nano):

  • VIM ( https://bioinformatics.uconn.edu/vim-guide/ ; https://missing.csail.mit.edu/2020/editors/)

  • Nano (https://engineering.purdue.edu/ECN/Support/KB/Docs/BasictutorialforNanou ; https://www.howtogeek.com/howto/42980/the-beginners-guide-to-nano-the-linux-command-line-text-editor/ )

• Have an HPC account on QUT’s lyra. Apply for a new HPC account here.

Install nextflow: Nextflow nf nextflow quick start

Database

Custom virus database, please do not distribute to third parties. Location:

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makeblastdb -in test.fasta -parse_seqids -dbtype nucl

Method

We will use two nextflow pipelines to process the Virome data, initially, we run trimgalore to filter out poor quality reads/bases and remove adapter sequences. Then we run VirReport to assess the presence of viruses and viroids.

1) Quality Control of Raw Files

First generate an ‘index.csv’ file that contains the Sample ID and path to the raw data file:

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qstat -u USERNAME

2) Diagnosis of plant viruses and viroids

Installing VirReport

The open-source VirReport code is available at https://github.com/eresearchqut/VirReport

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Note: the above command will store a cached copy of VirReport at '$HOME/.nextflow/NXF_SINGULARITY_CACHEDIR'

Running VirReport

1. Sample index file

To run VirReport it is required to create an 'index_samples.csv` that specifies the sample ID, path to raw data, minimal length, and the maximum length of reads to be used for diagnosis. For example:

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You can modify the above template with your own samples. Note, the files above can be the trimgalore processed files.

sampleid,samplepath,minlen,maxlen
CB,/work/img/test/trimgalore/results/Trim_Galore/CB_trimmed.fq.gz,21,22
CM,/work/img/test/trimgalore/results/Trim_Galore/CM_trimmed.fq.gz,21,22
CP,/work/img/test/trimgalore/results/Trim_Galore/CP_trimmed.fq.gz,21,22
TB1,/work/img/test/trimgalore/results/Trim_Galore/TB1_trimmed.fq.gz,21,22
TBG,/work/img/test/trimgalore/results/Trim_Galore/TBG_trimmed.fq.gz,21,22
TM,/work/img/test/trimgalore/results/Trim_Galore/TM_trimmed.fq.gz,21,22
TPS,/work/img/test/trimgalore/results/Trim_Galore/TPS_trimmed.fq.gz,21,22
TP,/work/img/test/trimgalore/results/Trim_Galore/TP_trimmed.fq.gz,21,22
TR1,/work/img/test/trimgalore/results/Trim_Galore/TR1_trimmed.fq.gz,21,22
TR2,/work/img/test/trimgalore/results/Trim_Galore/TR2_trimmed.fq.gz,21,22

2. Run VirReport using a PBS Pro script

Define nextflow configurations if different from provided template:

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