Page Comparison

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)

...

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:

sampleId,read1
CB,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/CB_H52LJDRX2_TCATGCGT_L001_R1.fastq.gz
CM,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/CM_H52LJDRX2_CTGCATCA_L001_R1.fastq.gz
CP,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/CP_H52LJDRX2_TCAGACTT_L001_R1.fastq.gz
TB1,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/TB1_H52LJDRX2_TCACTACG_L001_R1.fastq.gz
TBG,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/TBG_H52LJDRX2_CTTCACGA_L001_R1.fastq.gz
TM,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/TM_H52LJDRX2_CGTTCTGC_L001_R1.fastq.gz
TP,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/TP_H52LJDRX2_AAGTTATC_L001_R1.fastq.gz
TPS,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/TPS_H52LJDRX2_CTTCTTAA_L001_R1.fastq.gz
TR1,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/TR1_H52LJDRX2_TCAGTGAG_L001_R1.fastq.gz
TR2,/work/img/raw_data/AGRF_CAGRF22029755_H52LJDRX2/TR2_H52LJDRX2_TGACCGCG_L001_R1.fastq.gz

Create a PBS Pro submission script:

#!/bin/bash -l
#PBS -N nftrimgalore 
#PBS -l walltime=24:00:00
#PBS -l select=1:ncpus=1:mem=5gb

cd $PBS_O_WORKDIR
NXF_OPTS='-Xms1g -Xmx4g'
module load java

#run netflow pipeline
nextflow run trimgalore --indexfile index.csv --singleEnd --trim_qual 30

Submit the job to the HPC scheduler:

qsub launch.pbs

Check progress of the job:

qjobs

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

...

At the HPC, run the following command to get a copy of

...

Get a copy of the toolthe source code:

git clone https://github.com/eresearchqut/VirReport.git

Alternatively, run the following command to fetch and also test VirReport:

nextflow run eresearchqut/VirReport -profile singularity --indexfile index_example.csv

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:

...

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

2. Run VirReport

...

An alternative is to clone a copy of the VirReport (above) run the following command that will both download VirReport tool and also run a test:

...

using a PBS Pro script