How to perform a PikaVirus Service

This is a brief tutorial on how to perform a Pikavirus Service as a member of the ISCIII's Bioinformatics Unit! PikaVirus is a associated with Viral: Detection and characterization of viral genomes within metagenomic data service from service catalog.

PikaVirus is a bioinformatics best-practise analysis pipeline for metagenomic analysis following a new approach, based on eliminatory k-mer analysis, followed by assembly and posterior contig-binning. This service will allow us to identify the viral species present in a sample.

Let's get started with the service. When performing a PikaVirus service, remember that the typical acronym is VIRAL-DISCOVERY, but this may differ depending on the service.

First of all, follow the first steps to create a service. Once the new-service is finished, you'll have a new folder with 6 folders: ANALYSIS, DOC, RAW, REFERENCES, RESULTS and TMP (as explained here). We should check the following folders before going any further:

• DOC: This folder should contains a hpc_slurm_pikavirus.config file with the specific configuration for nf-core/mag in our HPC and some custom parameters like databases paths.
• RAW: Check that the number of files contained within the RAW folder is equal to the number of samples specified in iskyLIMS x 2, in the case that they are paired-end reads.

If everything is OK, we can get into the ANALYSIS folder and we'll find the following items inside:

• lablog_pikavirus: an executable file that creates the 00-reads folder, moves inside, creates symbolic links to the reads renaming them and renames the ANALYSIS01_PIKAVIRUS folder.
• samples_id.txt: a .txt file containing all the sample names, one per line, so there will be as many lines as samples associated with our service.
• ANALYSIS01_PIKAVIRUS: Folder with the main PikaVirus analysis files.

Now we can execute the lablog:

bash lablog_pikavirus

[!WARNING] If PikaVirus is not the only analysis in your service, don't forget to run the other lablogs before the next steps.

After executing this file, if everything is OK, we can now proceed with the next BU-ISCIII tool: scratch as explained here

Once this function is finished, we should go into the scratch_tmp folder and the specific ANALYSIS01_PIKAVIRUS folder associated with our service. Once we're inside, we will see the following folders/files:

• lablog: will create symbolic links to 00-reads and the samples_id.txt file, creates pikavirus.sbatch, _01_nf_pikavirus.sh and samplesheet.csv files.

Let's execute the lablog file:

bash lablog

Now, we should check we've loaded all the needed dependencies and perform the metagenomic analysis:

module load Nextflow/21.10.6 singularity
bash _01_nf_pikavirus.sh

After this, the analysis will start, and we'll be able to check the status of the process with:

tail -f DATE_pikavirus01.log

Once checked everything has finished OK in the DATE_pikavirus01.log, within the DATE_ANALYSIS01_PIKAVIRUS folder, we'll have the following content:

• 01-PikaVirus-results/: Results of the PikaVirus pipeline.
  • multiqc_report.html: MultiQC's HTML report of the PikaVirus pipeline.
  • mash_results: Mash results for each sample, we would need to check them if there is any problem with the hits.
  • <sample_name>: Results specific for the sample (quality control, multiQC, coverage...)
  • <sample_name>_results.html: HTML results with the stats for the sample (hits found, coverage, stats...)
  • all_samples_virus_table.tsv: Table with the results for all the samples and the stats in .tsv format.

The file all_samples_virus_table.tsv usually contains some duplicated reads and phages. In order to clean the results in this table we should run:

grep -v 'genome' all_samples_virus_table.tsv | grep -v 'phage' > all_samples_virus_table_filtered.tsv

Then convert this table to .xlsx if this process is not included in the RESULTS's lablog.

In this service we should check:

• 01-PikaVirus-results/multiqc_report.html: FastQC/fastp reports to assess the good/bad quality of the reads, the presence of adaptaers, etc...
• all_samples_virus_table.tsv: Check that the species are the spected and that they contain enough coverage. Those virus covered to >50% at a depth of 10X, can be used as reference genome for nf-core/viralrecon for further analysis.

Once all the service results are revised in the scratch_tmp folder, you can continue with the next BU-ISCIII tool: finish as explained here.

When everything is propperly coppied into the SFTP folder, you can put the update in the update_servicios channel's Team Standup Bot, with the following template:

• SRVXXXXXX - VIRAL-DISCOVERYXXX :no_entry: = No empezado, parado o esperando :running: = Corriendo :mag: = En revisión :clipboard: = Finalizado copiandose a SFTP :white_check_mark: = Finalizado y copiado a sftp
  • Servicio: Viral: Detection and characterization of viral genomes within metagenomic data (PikaVirus)
  • Número de muestras: # Numero de muestras
  • Estado: No empezado/Corriendo/En revisión/Finalizado copiandose a SFTP/Finalizado y copiado a sftp
  • Instrumento y longitud: Ej.: NovaSeq (2x150)
  • Cantidad de lecturas: Ej.: 0.8M - 91.2M
  • Calidad general: Buena/Mala/Indicar incidencias específicas
  • Resultados generales: Breve resumen de los resultados obtenidos a grandes rasgos, describiendo los resultados más relevantes del all_samples_virus_table.tsv.
  • Resultados por muestra: Indicar solo los resultados ANOMALOS. Algunos ejemplos:
    • La muestra XXX contiene X virus (si es que no se espera ese virus en esa muestra)
    • La muestra XXX se ha secuenciado con mala calidad y ha perdido muchas lecturas en el preprocesamiento.
    • La muestra XXX no tiene lecturas suficientes para realizar el análisis.

When the revisors give the OK to the service results, you can finish the service with the next BU-ISCIII tool: delivery as explained here.