nf-core/rnafusion

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Introduction

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline.

The directories listed below will be created in the results directory after the pipeline has finished. All paths are relative to the top-level results directory.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

• Download and build references - Build references needed to run the rest of the pipeline
• STAR - Alignment of FASTQ files
• Arriba - Arriba fusion detection
• STAR-fusion - STAR-fusion fusion detection
• StringTie - StringTie assembly
• FusionCatcher - Fusion catcher fusion detection
• CTAT-SPLICING - Detection and annotation of cancer splicing aberrations
• Samtools - SAM/BAM file manipulation
• Fusion-report - Summary of the findings of each tool and comparison to COSMIC, Mitelman, and FusionGDB2 databases
• FusionInspector - Supervised analysis of fusion predictions from fusion-report, recover and re-score evidence for such predictions
• Arriba visualisation - Arriba visualisation report for FusionInspector fusions
• Picard - Collect QC metrics
• FastQC - Raw read QC
• Salmon - Normalized gene expression calculation
• MultiQC - Aggregate report describing results and QC from the whole pipeline
• Pipeline information - Report metrics generated during the workflow execution

Download and build references

Main pipeline workflow

If no argument is specified here, the tool was used with default parameters.

Directory structure

{outdir}
├── agat
├── arriba
├── arriba_visualisation
├── ctatsplicing
├── fastp
├── fastqc
├── fastqc_for_fastp
├── fusioncatcher
├── fusioninspector
├── fusionreport
├── multiqc
├── picard
├── pipeline_info
├── salmon
├── star
├── starfusion
├── stringtie
└── vcf
.nextflow.log

If no parameters are specified, the default is applied.

Agat

Agat is to convert the GFF file to a TSV file, which is then used in the VCF creation.

Arriba

Arriba is used for i) detect gene fusions and ii) create a PDF report for the fusions found (visualisation):

Detection

Visualisation

The visualisation displays the fusions that fusioninspector outputs. That means that fusions from all callers are aggregated (by fusion-report) and then analyzed through fusioninspector (Note: Fusioninspecor contains a filtering step!).

CTAT-SPLICING

If --tools ctatsplicing is present, CTAT-SPLICING will detect cancer splicing aberrations.

CTAT-SPLICING detects and annotates of aberrant splicing isoforms in cancer. This is run on the input files for arriba and/or starfusion.

Fastp

If --tools fastp is present, fastp will filter low quality reads as well as bases at the 5’ and 3’ ends, trim adapters (automatically detected, but input with parameter --adapter_fasta is possible). 3’ trimming is also possible via parameter --trim_tail.

As fusioncatcher is especially sensitive to read length, 100 bp being the recommended length, an additional parameter --trim_tail_fusioncatcher triggers an extra fastp process with 3’ trimming of the length given, these triggered reads are then fed to fusioncatcher but the non-extra trimmed ones are still used for arriba and STAR-Fusion.

Fastp for fusioncatcher

If trim_tail_fusioncatcher has any value other than 0, fastp will be run again as above. This allows for additional trimming of read tails before running FusionCatcher. For example if reads are 150bp, using --trim_tail_fusioncatcher 50 will shorten reads to 100 bp by 50 bases from the 3′ end. 100 bp is the recommended read length to feed into FusionCatcher. The default for --trim_tail_fusioncatcher is 0 (no trimming).

FastQC

FastQC gives general quality metrics about your sequenced reads. It provides information about the quality score distribution across your reads, per base sequence content (%A/T/G/C), adapter contamination and overrepresented sequences. For further reading and documentation see the FastQC help pages.

FastQC for fastp

This directory contains FastQC reports for the fastp trimmed reads.

FusionCatcher

FusionCatcher searches for novel/known somatic fusion genes translocations, and chimeras in RNA-seq data. Possibility to use parameter --fusioncatcher_limitSjdbInsertNsj to modify limitSjdbInsertNsj.

FusionInspector

FusionInspector performs a validation of fusion transcript predictions. Possibility to use --fusioninspector_limitSjdbInsertNsj to set limitSjdbInsertNsj to anything other than the default 1000000.

Fusion-report

Please note that fusion-report is executed from fork https://github.com/Clinical-Genomics/fusion-report

Fusion-report is a tool for parsing outputs from fusion detection tools. The Fusion Indication Index is explained here: https://github.com/Clinical-Genomics/fusion-report/blob/master/docs/score.md. Summary:

The weights for databases are as follows:

• COSMIC (50)
• MITELMAN (50)
• FusionGDB2 (0)

The Fusion Indication Index FII is calculated using two components:

1. Tool Detection (50% of total FII)

   • Calculated as: (number of tools detecting the fusion) / (number of tools actually used)
   • This reflects how many of the active tools found the fusion

2. Database Hits (50% of total FII)

   • Based on database matches using weights above
   • Calculated as: (number of database hits) / (total possible database hits)

Final score = (0.5 × Tool Detection Score) + (0.5 × Database Hits Score)

MultiQC

Picard

Pipeline information

Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to troubleshoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage.

Salmon

Folder containing the quantification results

STAR

STAR is used to align FASTQ files to the genome reference.

Additionally, CRAM files can also be created when passing the --cram option. The CRAM conversion is done with a combination of samtools view and samtools index.

The STAR index is generated with --sjdbOverhang ${params.read_length - 1}, params.read_length default is 100.

STAR-fusion

StringTie

Vcf_collect