nf-eresearch/VirReport - Diagnosis of plant viruses and viroids using small RNA-seq data

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: Learning Resources: the Linux Command Line ; The Unix Shell: Summary and Setup )
Familiarity with one unix text editors (example Vi/Vim or Nano):
- VIM ( https://bioinformatics.uconn.edu/vim-guide/ ; Editors (Vim))
- Nano (Basic tutorial for Nano users ; 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 quick start

Database

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

/work/img/databases/

Creating a local blast database

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 the 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

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:

sampleid,samplepath,minlen,maxlen
MT212,/work/hia_mt18005/diagnostics/2021/14_RAMACIOTTI_LEL9742-LEL9751/results/06_usable_reads/MT212_21-22bp.fastq,21,22
MT213,/work/hia_mt18005/diagnostics/2021/14_RAMACIOTTI_LEL9742-LEL9751/results/06_usable_reads/MT213_21-22bp.fastq,21,22

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:

includeConfig 'conf/base.config'

params {
  outdir = 'results'
  indexfile = 'index.csv'
  blast_db_dir = '/lustre/work-lustre/hia_mt18005/blastDB/30112021'
  blast_local_db_path = '/work/img/databases/PVirDB/PVirDB_ver20211109.fasta'
  targets = false
  targets_file = 'Targetted_Viruses_Viroids.txt'
  help = false
  cap3_len = '20'
  orf_minsize = '150'
  orf_circ_minsize = '150'
  blastn_evalue = '0.0001'
  blastp_evalue = '0.0001'
  blastn_method = 'megablast'
  blastp = false
  spades = false
  spadeskmer = '9 11 13 15 17 19 21'
  blastlocaldb = false
  ictvinfo = 'ICTV_taxonomy_MinIdentity_Species.tsv'
  contamination_detection = false
  contamination_flag = '0.01'
  contamination_detection_method = 'FPKM'
}

process.container = "ghcr.io/eresearchqut/virreport:v1.0.0"

manifest {
  name          = "eresearchqut/VirReport"
  author        = "Roberto Barrero, Maely Gauthier, Desmond Schmidt, Craig Windell"
  defaultBranch = "main"
  description   = "VirReport is designed to help phytosanitary diagnostics of viruses and viroid pathogens in quarantine facilities. It takes small RNA-Seq samples as input."
  version       = "v1.0.0"
}

Prepare 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 eresearchqut/VirReport -profile singularity --indexfile index.csv