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eresearchqut/VirReport is a bioinformatics pipeline based upon the scientific workflow manager Nextflow. It was has been designed to help phytosanitary diagnostics of viruses and viroid pathogens in quarantine facilities. It takes small RNA-Seq fastq files as input. These can either be in raw format (currently only samples specifically prepared with the QIAGEN QIAseq miRNA library kit can be processed this way) or quality-filtered.

For target identification, VirReport uses an hybrid de novo assembly approach to build contigs that are then annotated using blast homology searches against either a virus database or/and a local copy of NCBI nr and nt databases.

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• Retain reads of a given length (21-22 nt long by default) from fastq file(s) provided in the index.csv file (READPROCESSING)

• De novo assembly using both Velvet and SPAdes. The contigs obtained are collapsed into scaffolds using cap3. By default, only contigs > 30 40 bp will be retained (DENOVO_ASSEMBLY)

• Run megablast homology search against either a local virus database or NCBI NT/NR databases:

• Searches against a local virus database:

  • Run megablast homology searches on de novo assembly against local virus and viroid database. Homology searches against blastn are also run in parallel for comparison with the megablast algorithm (BLAST_NTBLASTN_VIRAL_DB_CAP3)

  • Retain top megablast hit and restrict results to virus and viroid matches. Summarise results by grouping all the de novo contigs matching to the same viral hit and deriving the cumulative blast coverage and percent ID identity for each viral hit (FILTER_BLAST_NTBLASTN_VIRAL_DB_CAP3)

  • Align reads to top hit, derive coverage statistics , and consensus sequence and VCF matching to top blast hit (FILTER_BLAST_NT_VIRAL_DB_CAP3, COVSTATS_VIRAL_DB)

  • Run tblastn homolgy search on predicted ORF >= 90 bp derived using getORF (TBLASTN_VIRAL_DB)

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• Searches against local NCBI NT and NR databases:

  • Retain top 5 megablast hits and restrict results to virus and viroid matches. Summarise results by grouping all the de novo contigs matching to the same viral hit and deriving the cumulative blast coverage and percent ID for each viral hit (BLASTN_NT_CAP3)

  • Align reads to top hit, derive coverage statistics, consensus sequence and VCF matching to top blast hits (COVSTATS_NT)

  • Run blastx homolgy search on contigs >= 90 bp long for which no match was obtained in the megablast search. Summarise the blastx results and restrict to virus and viroid matches (BLASTX)

• The pipeline can perform additional optional steps, which include:

• A quality filtering step on raw fastq files (currently the workflow only processes samples prepared using QIAGEN QIAseq miRNA library kit). After performing quality filtering (FASTQC_RAW, ADAPTER_AND_QUAL_TRIMMING, QCQUAL_POSTTRIMING_QUALAND_TRIMMINGQC, DERIVE_USABLE_READS). the The pipeline will also derive a qc report (QCREPORT). An RNA souce profile can also be included as part of this the quality filtering step (RNA_SOURCE_PROFILE, RNA_SOURCE_PROFILE_REPORT)

• VirusDetect version 1.8 can also be run in parallel. A summary of the top virus/viroid blastn hits will be separately output (VIRUS_DETECT, VIRUS_IDENTIFY, VIRUS_DETECT_BLASTN_SUMMARY, VIRUS_DETECT_BLASTN_SUMMARY_FILTERED)

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This link specifically describes the steps to take to load Java and install Nextflow on our local HPC at QUT (Lyra): Nextflow

3B. Installing a suitable environment management system

To run the VirReport pipeline, you will need to install a suitable environment management system such as Docker, Singularity or Conda to suit your environment.

We use conda or miniconda on our HPCrecommnend using Singularity.

3C. Installing VirReport

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nextflow run eresearchqut/VirReport -profile {docker, singularity or conda}

On our HPC, specify singularity We recommend to use Singularity as the profile.

Cached environment will be built in your home directory under the cached singularity directory. This step will take some time the first time you run the pipeline.

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• By default, the pipeline is set to run homology blast searches against a local plant virus/viroid database (this is set in the nextflow.config file with parameter --virreport_viral_db = true. You will need to provide this database to run the pipeline. You can either provide your own or use a A curated database is provided at https://github.com/maelyg/PVirDB.git. Ensure you use NCBI BLAST+ makeblastdb to create the database. For instance, to set up this database, you would take the following steps:

  Code Block
  git clone https://github.com/maelyg/PVirDB.git cd PVirDB gunzip PVirDB_v1.fasta.gz makeblastdb -in PVirDB_v1.fasta -parse_seqids -dbtype nucl

  Then specify the full path to the database files including the prefix in the nextflow.config file. For example:

  Code Block
  params { blast_local_db_path = '/path_to_viral_DB/viral_DB_name' }

• If you also want to run homology searches against public NCBI databases, you need to set the parameter virreport_ncbi in the nextflow.config file to true:

  Code Block
  params { virreport_ncbi = true }

  or add it in your nextflow command:

  Code Block
  nextflow run eresearchqut/VirReport -profile {docker, singularity or conda} --virreport_ncbi

  Download these locally, following the detailed steps available at https://www.ncbi.nlm.nih.gov/books/NBK569850/ . Create a folder where you will store your NCBI databases. It is good practice to include the date of download. For instance:

  Code Block
  mkdir blastDB/30112021

  You will need to use the update_blastdb.pl script from the blast+ version used with the pipeline.
  For example:

  Code Block
  perl update_blastdb.pl --decompress nt [*] perl update_blastdb.pl --decompress nr [*] perl update_blastdb.pl taxdb tar -xzf taxdb.tar.gz

  Make sure the taxdb.btd and the taxdb.bti files are present in the same directory as your blast databases.
  Specify the path of your local NCBI blast nt and nr directories in the nextflow.config file.
  For instance:

  Code Block
  params { blast_db_dir = '/work/hia_mt18005_db/blastDB/20220408' }

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• By default the pipeline expects a single quality-filtered fastq file per sample.

• If you want to provide raw fastq files, samples have to be specifically prepared with the QIAGEN QIAseq miRNA library kit. If you want to run the initial quality filtering step on your raw fastq files, you will need to set the --qualityfilter paramater to true in the config.file and specify the path to the directory which holds the required bowtie indices (using the --bowtie_db_dir parameter) to: 1) filter non-informative reads (using the blacklist bowtie indices for the DERIVE_USABLE_READS process) and 2) optionally derive the origin of the filtered reads obtained (RNA_SOURCE_PROFILE process).

  

  The required fasta files are available at https://github.com/maelyg/bowtie_indices.git and bowtie indices can be built from these using the command:

  Code Block
  git clone https://github.com/maelyg/bowtie_indices.git gunzip blacklist_v2.fasta.gz #you might need to activate your environment cached in either your conda or singularity environment in order to run bowtie #for example conda activate /path_to_cached_environment/virreport-77d02f3abe1d8ba5f8dfdff194142de9 #then run the bowtie command bowtie-build -f blacklist_v2.fasta blasklistblacklist

  The directory in which the bowtie indices are located will need to be specified in the nextflow.config file:

  Code Block
  params { bowtie_db_dir = '/path_to_bowtie_idx_directory' }

  If you are interested to derive an RNA source profile of your fastq files you will need to specify:

  Code Block
  params { rna_source_profile = true }

  And build the other indices from the fasta files included in https://github.com/maelyg/bowtie_indices.git (i.e. rRNA, plant_tRNA, plant_noncoding, plant_pt_mt_other_genes, artefacts, plant_ miRNA, virus).

  The quality filtering step will create the 00_quality_filtering folder under the results folder:

  Code Block

  results/ ├── 00_quality_filtering └── sample_name │ ├── sample_name_18-25nt_cutadapt.log │ ├── sample_name_fastqc.html │ ├── sample_name_fastqc.zip │ ├── sample_name_21-22nt_cutadapt.log │ ├── sample_name_21-22nt.fastq.gz │ ├── sample_name_24nt_cutadapt.log │ ├── sample_name_blacklist_filter.log │ ├── sample_name_fastp.html │ ├── sample_name_fastp.json │ ├── sample_name_qual_filtering_cutadapt.log │ ├── sample_name_quality_trimmed_fastqc.html │ ├── sample_name_quality_trimmed_fastqc.zip │ ├── sample_name_quality_trimmed.fastq.gz │ ├── sample_name_read_length_dist.pdf │ ├── sample_name_read_length_dist.txt │ ├── sample_name_truseq_adapter_cutadapt.log │ └── sample_name_umi_tools.log └── qc_report ├── read_origin_counts.txt ├── read_origin_detailed_pc.txt ├── read_origin_pc_summary.txt ├── run_qc_report.txt └── run_read_size_distribution.pdf

  If your sequencing run was split on multiple lanes, you might have several raw fastq files per sample, and you can directly feed these to the pipeline and specify the --merge-lane parameter. The fastq files will be collapsed to one fastq file before performing downstream analysis. The sample name used will be the sampleid provided in the index.csv file. In the example below 2 fastq files were generated for 1 sample named CT103:

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nextflow run eresearchqut/VirReport -profile singularity -resume --indexfile index.csv \
                                    --merge_lane --qualityfilter --rna_source_profile \
                                    --bowtie_db_dir /path_to_bowtie_indices \
                                    --virreport_ncbi --blast_viral_db_pathdir /path_to_ncbi_databases

Deduplicate reads using unique molecular identifiers and mapping coordinates

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If you want to derive a summary of detections for all the samples included in the index file, specify the --contaminationdetecion_detectionreporting_viral_db or the --contaminationdetection_detectionreporting_ncbint option. This will create a summary text file under the Summary tab with a column called contamination_flag

Running the Velvet pipeline

VirusDetect version 1.8 can also be run in parallel.

See http://virusdetect.feilab.net/cgi-bin/virusdetect/index.cgi for details about this separate pipeline.

Example of PBS script to run on an HPC with torque batch system

Make sure to either specify the full path to your index.csv file in the PBS script or place a copy of the index.csv file in the folder you will run the PBS script in.

The PBS script example below (VirReport_nextflow.sh) will run on raw fastq files that will need to be merged and then quality filtered.

We are also asking to run a process that will derive an RNA source profile for each samples during the quality filtering step.

Homology searches will be run against NCBI and the PVirDB. The pipeline will also run VirusDetect in parallel.

Finally we will want the reads to be de-duplicated after mapping.

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With the contamination flag, the assumption is that if a pest is present at high titer in a given sample and detection of reads matching to this pathogen in other samples occur at a significantly lower abundance, there is a risk that this lower signal is due to contamination (e.g. index hopping from high-titer sample). We first calculate the maximum FPKM value recorded for each virus and viroid identified on a run. If for a given virus, the FPKM value reported for a sample represented less than a percentage of this maximum FPKM value, it is then flagged as a contamination event. We apply a 1% threshold value as default. This is just indicative and method cannot discriminate between false positives and viruses present at very low titer in a plant. It is then recommended to compare the sequences obtained, check the SNPs and validate through independent method.

Running in diagnostic mode (SSG team only internal use)

If you want to run VirReport in diagnostics mode (--diagno), the pipeline will also add an evidence category (ie KNOWN, KNOWN_FRAGMENT and CANDIDATE_NOVEL) to each detection based on av-pident and % bases 10X.

If you are running homology searches against the NCBI NT database, you will also need to provide a list of pests of interest in the Targetted_Virus_Viroid.txt file located in the bin folder. If some of the detections match to this pest list, they will be categorised as Quarantinable versus Higher_plant_viruses in the final summary.

Finally, if sample information is provided (--sampleinfo --sampleinfo_path /path/to/sampleinfo.txt and --samplesheet_path /path/to/Sample_Sheet.csv), this will be added to the final summary.

Sampleinfo.txt file example:

Sample	PEQ_index_number	LIMS_ID_RAMACIOTTI	Host_species	Host_common_name	Plant_tissue_collected
MT498	P30	ELL110002A1	Allium sativum	Garlic	50 mg leaf

Running VirusDetect

VirusDetect version 1.8 can also be run in parallel.

See http://virusdetect.feilab.net/cgi-bin/virusdetect/index.cgi for details about this separate pipeline.

Example of PBS script to run on an HPC with torque batch system

Make sure to either specify the full path to your index.csv file in the PBS script or place a copy of the index.csv file in the folder you will run the PBS script in.

Example 1:

The PBS script example below (VirReport_nextflow.sh) will run on raw fastq files that will need to be merged and then quality filtered.

We are also asking to run a process that will derive an RNA source profile for each samples during the quality filtering step.

Blastn (using the megablast algorithm) and tblastn homology searches will be run against the PVirDB.

Finally we will want the reads to be de-duplicated after mapping.

#!/bin/bash -l
#PBS -N VirReport
#PBS -l select=1:ncpus=2:mem=8gb
#PBS -l walltime=05:00:00


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

nextflow run eresearchqut/VirReport -profile singularity -resume --indexfile index.csv \
                                    --merge_lane --qualityfilter --rna_source_profile \
                                    --bowtie_db_dir /path_to_bowtie_indices \
                                    --dedup \
                                    --virreport_viral_db --blast_viral_db_path /path_to_local_viral_database \
                                    --detecion_reporting_viral_db

Example 2:

In the PBS job below, blastn (using the megablast algorithm) and blastx homology searches will be run against NCBI NR and NT respectively.

#!/bin/bash -l
#PBS -N VirReport
#PBS -l select=1:ncpus=2:mem=8gb
#PBS -l walltime=05:00:00


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

nextflow run eresearchqut/VirReport -profile singularity -resume --indexfile index.csv \
                                    --merge_lane --qualityfilter --rna_source_profile \
                                    --bowtie_db_dir /path_to_bowtie_indices \
                                    --dedup \
                                    --virreport_ncbi --blast_viral_db_path /path_to_ncbi_databases \
                                    --detection_reporting_nt

Example 3:

In the PBS job below, homology searches will be run against NCBI and the PVirDB. The pipeline will also run VirusDetect in parallel.

#!/bin/bash -l
#PBS -N VirReport
#PBS -l select=1:ncpus=2:mem=8gb
#PBS -l walltime=05:00:00


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

nextflow run eresearchqut/VirReport -profile singularity -resume --indexfile index.csv \
                                    --merge_lane --qualityfilter --rna_source_profile \
                                    --bowtie_db_dir /path_to_bowtie_indices \
                                    --dedup \
                                    --virreport_ncbi --blast_viral_db_path /path_to_ncbi_databases --detecion_reporting_viral_nt \
                                    --virreport_viral_db --blast_viral_db_path /path_to_local_viral_database --detection_reporting_viral_db \
                                    --virusdetect --virusdetect_db_path
                                    
                                    
                                    
sampleid,samplepath
MT500,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/ELL11002/ELL11002A3/MT500_S3_L001_R1_001.fastq.gz
MT500,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/ELL11002/ELL11002A3/MT500_S3_L002_R1_001.fastq.gz
MT502,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/ELL11002/ELL11002A5/MT502_S5_L001_R1_001.fastq.gz
MT502,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/ELL11002/ELL11002A5/MT502_S5_L002_R1_001.fastq.gz
MT512,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/LEL11109/LEL11109A1/Fn1_S25_L001_R1_001.fastq.gz
MT512,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/LEL11109/LEL11109A1/Fn1_S25_L002_R1_001.fastq.gz
MT524,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/LEL11109/LEL11109A13/FraD3_S28_L001_R1_001.fastq.gz
MT524,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/LEL11109/LEL11109A13/FraD3_S28_L002_R1_001.fastq.gz
CT113,/work/hia_mt18005/raw_data/20220629_RAMACIOTTI_DES10730/DES10730A20/CT_113_S20_L001_R1_001.fastq.gz
CT113,/work/hia_mt18005/raw_data/20220629_RAMACIOTTI_DES10730/DES10730A20/CT_113_S20_L002_R1_001.fastq.gz
CT140,/work/hia_mt18005/raw_data/20220629_RAMACIOTTI_DES10730/DES10730A47/CT_140_S47_L001_R1_001.fastq.gz
CT140,/work/hia_mt18005/raw_data/20220629_RAMACIOTTI_DES10730/DES10730A47/CT_140_S47_L002_R1_001.fastq.gz
MT515,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/LEL11109/LEL11109A4/Cn1_S19_L001_R1_001.fastq.gz
MT515,/work/hia_mt18005/raw_data/20220915_RAMACIOTTI_ELL11002_LEL11109/LEL11109/LEL11109A4/Cn1_S19_L002_R1_001.fastq.gz
MT005,/work/hia_mt18005/raw_data/20210618_RAMACIOTTI_ELL9278/ELL9278/ELL9278A04/MT005_S4_L001_R1_001.fastq.gz
MT005,/work/hia_mt18005/raw_data/20210618_RAMACIOTTI_ELL9278/ELL9278/ELL9278A04/MT005_S4_L002_R1_001.fastq.gz
2223PEQ041,/work/hia_mt18005/raw_data/20221018_RAMACIOTTI_LEL11294/LEL11294/LEL11294A15/2223PEQ041_S15_L001_R1_001.fastq.gz
2223PEQ041,/work/hia_mt18005/raw_data/20221018_RAMACIOTTI_LEL11294/LEL11294/LEL11294A15/2223PEQ041_S15_L002_R1_001.fastq.gz
MT447,/work/hia_mt18005/raw_data/20220218_RAMACIOTTI_LEL10024/MT447_S40_L001_R1_001.fastq.gz
MT447,/work/hia_mt18005/raw_data/20220218_RAMACIOTTI_LEL10024/MT447_S40_L002_R1_001.fastq.gz
MT449,/work/hia_mt18005/raw_data/20220218_RAMACIOTTI_LEL10024/MT449_S33_L001_R1_001.fastq.gz
MT449,/work/hia_mt18005/raw_data/20220218_RAMACIOTTI_LEL10024/MT449_S33_L002_R1_001.fastq.gz
2223PEQ012,/work/hia_mt18005/raw_data/20221018_RAMACIOTTI_LEL11291/LEL11291/LEL11291A12/2223PEQ012_S12_L001_R1_001.fastq.gz
2223PEQ012,/work/hia_mt18005/raw_data/20221018_RAMACIOTTI_LEL11291/LEL11291/LEL11291A12/2223PEQ012_S12_L002_R1_001.fastq.gz

Submit your job using the qsub command:

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Alternatively use the following command to check on the jobs you are running.:

qjobs

You can also check the .nextflow.log file for details on progress.

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• assembly: results associated with de novo assembly

• blastn: megablast results (NCBI NT or viral database PVirDB)

• blastx: blastx results against NR

• tblastn: tblastn results against viral database PVirDB

• alignments: alignment against top reference hit and associated statistic derivation

• Summary

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Definitions of terms used in summary report: 

• sacc  Accession number of best homology match recovered

• av-pident  Average per cent identity of all de novo assembled contigs to the same top reference hit

• Mean read depth  The mean coverage in bases to the genome/sequence of the best homology match

• Dedup read count  Read counts after PCR duplicates sharing UMIs are collapsed

• Dup %  Duplication rate detected using UMIs

• FPKM:  Fragments Per Kilobase of transcript, per Million mapped reads is a normalised unit of

•   transcript expression. It scales by transcript length to compensate for the fact that most

•   RNA-seq protocols will generate more sequencing reads from longer RNA molecules

•   [deduplicated read count x 10^3 x 10^6]/[total quality filtered reads x genome length]

• % bases 5X  The fraction of bases that attained at least 5X sequence coverage

• % bases 10X  The fraction of bases that attained at least 10X sequence coverage

• Contamination flag  Assumption: If a pest is present at high titer in a given sample and detection of reads matching to this pathogen in other samples occur at a significantly lower abundance, there is a risk that this lower signal is due to contamination (e.g. index hopping from high-titer sample). We first calculate the maximum FPKM value recorded for each virus and viroid identified on a run. If for a given virus, the FPKM value reported for a sample represented less than a percentage of this maximum FPKM value, it is then flagged as a contamination event. We apply 0.1% threshold value as default. This is just indicative and method cannot discriminate between false positives and viruses present at very low titer in a plant. It is then recommended to compare the sequences obtained, check the SNPs and validate through independent methodde novo assembly

• blastn: megablast results (NCBI NT or viral database PVirDB)

• blastx: blastx results against NR

• tblastn: tblastn results against viral database PVirDB

• alignments: alignment against top reference hit and associated statistic derivation

• Summary

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Definitions of terms used in summary report: 

• sacc  Accession number of best homology match recovered

• av-pident  Average per cent identity of all de novo assembled contigs to the same top reference hit

• Mean read depth  The mean coverage in bases to the genome/sequence of the best homology match

• Dedup read count  Read counts after PCR duplicates sharing UMIs are collapsed

• Dup %  Duplication rate detected using UMIs

• FPKM:  Fragments Per Kilobase of transcript, per Million mapped reads is a normalised unit of transcript expression. It scales by transcript length to compensate for the fact that most RNA-seq protocols will generate more sequencing reads from longer RNA molecules. The formula is: [deduplicated read count x 10^3 x 10^6]/[total quality filtered reads x genome length]

• % bases 5X  The fraction of bases that attained at least 5X sequence coverage

• % bases 10X  The fraction of bases that attained at least 10X sequence coverage

• Contamination flag.