VSD-1.0 (Virus Surveillance and Diagnosis)

General information

The viral surveillance and diagnosis (VSD) bioinformatics toolkit is a pipeline based on the scientific workflow manager Nextflow.

It is designed to help phytosanitary diagnostics of viruses and viroid pathogens in quarantine facilities. It takes small RNA-Seq samples as input.

Installation

Install Nextflow

The VSD workflow requires Nextflow to be installed in your account on the HPC. Find details on how to install and test Nextflow here prepare a nextflow.config file and run a PBS pro submission script for Nextflow pipelines.

Pull the git repo using:

Install conda3 or miniconda3

Installing on Linux — conda 24.9.3.dev45 documentation

All the required modules are included in the environment.yml file in the pipeline base directory and shows all the tools used in the pipeline:

Install a local NCBI blast directory (NT and NR)

Find detailed infor on how to download these databases at https://www.ncbi.nlm.nih.gov/books/NBK569850/

Make sure the taxdb.btd and the taxdb.bti files are also present in the directory.

Create a folder where you will store your NCBI database including the date of download. For instance:

Run the following PBS script in the newly created folder. Use the update_blastdb.pl script from the blast+ version you will use with your pipeline.

The VSD workflow

The VSD workflow will perform the following steps by default:

• Retain reads of a given length (e.g. 21-22 or 24 nt long) from fastq file(s) provided in index.csv file (readprocessing)

• De novo assembly using kmer 15 and coverage 3 (velvet) -

• Collapse contigs into scaffolds (min length 20) (cap3)

• Run megablast homology search against NCBI NT database (megablast_nt_velvet)

• Summarise megablast results and restrict to virus and viroid matches (BlastTools_megablast_velvet)

• Derive coverage statistics, consensus sequence and VCF matching to top blast hits (filter_n_cov)

A number of additional optional steps can be run:

• --blastp: Predict ORF from de novo assembly (derived with Velvet) and run blastP againts NCBI NR (getorf, blastp, blastpdbcmd, BlastToolsp) --blastp

• --contamination_detection: Run cross-sample contamination prediction (contamination_detection)

• --blastlocaldb: Run blastn and megablast homology search on de novo assembly (derived with Velvet) against local virus and viroid database (blast_nt_localdb_velvet, filter_blast_nt_localdb_velvet)

• --blastn: Run blastn homology search on de novo assembly (derived with Velvet) against local virus and viroid database (blastn_nt_velvet)

• --spades: Run SPAdes 3.14 de novo assembler and perform blastn homology analysis on the derived de novo contigs (spades, cap3_spades, megablast_nt_spades , BlastToolsn_megablast_spades)

A number of additional options are included:

• --targets: A text file with the taxonomy of the viruses/virioids of interest can be provided and only these will be retained in the megablast summary results derived at the filter_n_cov step.

• --spadeskmer specifies the range of kmers to use when running spades

• --cap3_len specifies the minimal length of contigs to retain after CAP3 scaffolding

• --blastn_evalue and --blastp_evalue specifies the evalue parameter to use during blast analyses

• --orf_minsize correspond to the minimal open reading frame getorf retains

To enable these options, they can either be included in the nextflow run command provided in the PBS script:

or update parameter to true in the nextflow.config file. For instance:

Preparing the data

Preparing an index.csv file

You need to create a TAB delimited text file that will be the input for the workflow. By default the pipeline will look for a file called “index.csv” in the base directory but you can specify any file name using the --indexfile [filename] in the nextflow run command. This text file requires the following columns (which needs to be included as a header): sampleid,samplepath,minlen,maxlen:

• sampleid will be the sample name that will be given to the files created by the pipeline

• samplepath is the full path to the quality filtered fastq files that the pipeline requires as starting input

• minlen and maxlen correspond to the read size that will be retained for downstream analyses.

An index_example.csv is included in the base directory:

You also need to provide the path of your NCBI blast directory in the nextflow.config file. For instance:

If you are interested to run a blast analysis against a local database, you also need to specify its path in the nextflow.config file. For example:

Running the pipeline

Finally you need to create a PBS script which includes your nextflow run command. An example of PBS script is included in the base directory and will run the pipeline with default steps:

This following PBS script will additionally run spades, the cross sample predictor and blast searches against a local database:

These jobs can be submitted using:

Monitoring the run

You can use the command

Alternatively use the following command:

To check on the jobs you are running. Nextflow will launch additional jobs during the run.

You can also check the .nextflow.log file for details on what is going on.

Finally, if you have configured the connection to the NFTower you can logon and check your run.

Outputs

Under the results folder, the pipeline will populate outputs under separate folders for each step. These will be stored in subfolders for each sample:

results → 01_read_size_selection → sample1

etc…

The folders are structures as follows (examples of outputs are provided in italics):

• 01_read_size_selection (cutadapt log file and fastq file including reads only matching the size specified in the index.csv file) MT020_21-22nt_cutadapt.log & MT020_21-22nt.fastq

• 02_velvet (velvet results and the fasta file which includes the velvet assembled contigs MT020_velvet_assembly_21-22nt.fasta

• 02a_spades (if spades is additionally run)

• 03_cap3 (fasta file of the scaffolds produced by CAP3 as well as the singletons) MT020_velvet_cap3_21-22nt_rename.fasta

• 04_blastn (all blastn results, filtered results limited to only viruses and viroid top 5 hit matches and their taxonomy) MT020_velvet_21-22nt_megablast_vs_NT.bls, MT020_velvet_21-22nt_megablast_vs_NT_top5Hits.txt, MT020_velvet_21-22nt_megablast_vs_NT_top5Hits_virus_viroids_final.txt MT020_velvet_21-22nt_megablast_vs_NT_top5Hits_virus_viroids_seq_ids_taxonomy.txt

• 05_blastoutputs (BlastTools.jar summary output which clusters all the contigs matching to a specific hit. summary_MT029_velvet_21-22nt_megablast_vs_NT_top5Hits_virus_viroids_final.txt

• 06_blastp (blastp outputs) MT020_velvet_21-22nt_getorf.min50aa.fasta, MT020_velvet_21-22nt_getorf.min50aa_blastp_vs_NR_out_virus_viroid.txt

• 07_filternstats (filtered blast summary with various coverage statistics for each virus and viroid hit, and associated consensus fasta file and vcf file) MT020_21-22nt_top_scoring_targets_with_cov_stats.txt, MT020_21-22nt_MK929590_Peach_latent_mosaic_viroid.consensus.fasta, MT020_21-22nt_MK929590_Peach_latent_mosaic_viroid_sequence_variants.vcf.gz

• 08_summary (summary of results for all samples included in the index.csv file. This includes a cross-contamination prediction) run_top_scoring_targets_with_cov_stats_with_cont_flag_21-22nt_0.01.txt

Future potential additional features:

• Include a deduplication step for fastq files that have UMIs incorporated

• Incorporate the fastq file initial filtering steps from sRNAqc as option

• Work on final summary report

• Add coverage statistics and cross contamination flag logic to local db blast results

• Incorporate VirusDetect in the pipeline and derive a summary of results from both pipelines

• Perform automatically 21-22nt and 24nt analyses by default