RNAsplicing-nf

Nextflow pipeline to perform RNA splicing analyses

Description

Runs the SUPPA2 software after recommended data processing of fastq files.

Dependencies

1. This pipeline is based on nextflow. As we have several nextflow pipelines, we have centralized the common information in the IARC-nf repository. Please read it carefully as it contains essential information for the installation, basic usage and configuration of nextflow and our pipelines.
2. External software:

• trimgalore
• salmon
• SUPPA2

You can avoid installing all the external software by only installing Docker. See the IARC-nf repository for more information.

Input

Type	Description
input_folder	Folder containing fastq files to be aligned
input_file	Input file (tab-separated values) with 4 columns: ID (sample name), RG (read group), pair1 (first fastq pair file), and pair2 (second fastq pair file)

Parameters

• Mandatory

Name	Example value	Description
index	salmon_index	Reference fasta file (with index) for splice junction trimming and base recalibration
gtf	GRCh38_gencode_annotation.gtf	Path to annotation gtf file

• Optional

Name	Default value	Description
output_folder	RNAsplicing-nf_output	Output folder'
cpu	4	Number of cpu used by salmon (default: 4).'
mem	10	Size of memory used for mapping (in GB) '
fastq_ext	fq.gz	Extension of fastq files '
suffix1	1	Suffix of fastq files 1'
suffix2	2	Suffix of fastq files 2'
mem_QC	2	Size of memory used for QC and cutadapt (in GB)'
cpu_trim	15	Number of cpu used by cutadapt '
index	salmon_index	Path to salmon index folder'
suppa_folder	SUPPA	Path to SUPPA folder (cloned from github) containing suppa.py and folders scripts.'
ioe	null	Path to SUPPA ioe file. If not specified, will generate the file.'

Usage

nextflow run iarcbioinfo/RNAsplicing-nf --input_file input.tsv --index salmon_index --gtf annot.gtf 

Output

Type	Description
QC/adapter_trimming	Fastqc reports from trim_galore
quantification	Transcript expression quantification from salmon
results/*ioe and ioi files	SUPPA2 ioe and ioi files used for quantification of psi by events and isoforms
results/psiPerEvent	Files with psi per event for each sample
results/psiPerIsoform	Files with psi per isoform for each sample

Detailed description

Nextflow pipeline that implements the SUPPA2 tutorial, first running salmon for quantification, then postprocessing the salmon outputs, creating the SUPPA2 ioe and ioi files if necessary, and computing psi per event and isoform for each sample.

Directed Acyclic Graph

Contributions

Name	Email	Description
Nicolas Alcala*	alcalan@iarc.who.int	Developer to contact for support
Ricardo Blazquez Encinas-Rey		Developer

References

• Trincado JL, Entizne JC, Hysenaj G, Singh B, Skalic M, Elliott DJ, Eyras E. SUPPA2: fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions. Genome Biol. 2018 Mar 23;19(1):40.
• Alamancos GP, Pagès A, Trincado JL, Bellora N, Eyras E. Leveraging transcript quantification for fast computation of alternative splicing profiles. RNA. 2015 Sep;21(9):1521-31.