IPSA-nf

An Integrative Pipeline for Splicing Analyses (IPSA) written in the Nextflow DSL.

The pipeline allows to perform the following splicing analyses:

• Quantification of splice junctions and splice sites
• Calculation of exon-centric and intron-centric splicing metrics
• Identification of micro-exons

Quickstart

Install nextflow with the following command:

curl -fsSL get.nextflow.io | bash

Pull the docker image:

docker pull guigolab/ipsa-nf@sha256:29750072f2b42ee8ea094a331f53bc5906183591a71ed26619ea76b12b6be3ed

Launch the test pipeline with the following command:

./nextflow run guigolab/ipsa-nf

Pipeline usage

Launching the pipeline with the --help parameter shows the help message:

nextflow run ipsa-nf --help

N E X T F L O W  ~  version 0.24.4
Launching `guigolab/ipsa-nf` [dreamy_aryabhata] - revision: v4.0
I P S A ~ Integrative Pipeline for Splicing Analyses
----------------------------------------------------
Run IPSA on a set of data.
Usage: 
    ipsa-nf [options]
Options:
--index INDEX_FILE        the index file in TSV format
--genome GENOME_FILE      the genome file in FASTA format
--annot ANNOTATION_FILE   the annotation file in gtf format
--merge MERGE             prefix for merged output files (default: all)
--dir DIRECTORY           the output directory
--sjcount-params PARAMS   additional `sjcount` paramters
--margin MARGIN           margin for aggregate (default: 5)
--mincount MIN_COUNT      minimum number of counts for denominators when calculationg fractions (default: 10)
--deltaSS DELTA           distance threshold for splice sites (default: 10)
--entropy ENTROPY         entropy lower threshold (default: 1.5)
--status STATUS           annotation status lower threshold (default: 0)
--microexons              include microexons, default=false

Input format

IPSA-nf takes as input a tab separated file containing information about the input data. The file must be specified using the --index parameter. The format of the index file is as follows:

1. sample identifier
2. path to the bam file to be processed
3. library type:
   • Single-End
   • Paired-End
4. strandnesss of the data:
   • NONE for unstranded
   • SENSE/ANTISENSE for Single-End stranded
   • MATE1_SENSE/MATE2_SENSE for Paired-End stranded

Here is an index file example:

E14_rep1    E14AlnRep1.sub.bam    Paired-End    MATE2_SENSE
E14_rep2    E14AlnRep2.sub.bam    Paired-End    MATE2_SENSE
E18_rep1    E18AlnRep1.sub.bam    Paired-End    MATE2_SENSE
E18_rep2    E18AlnRep2.sub.bam    Paired-End    MATE2_SENSE

Pipeline results

Analyses results are saved into the folder specified with the --dir parameter. By default it is the data directory within the current working folder.

Output files are organinzed into folders corresponding to the different pipeline endpoints:

• A01 - splice junctions and splice sites counts
• A02 - aggregated splice junctions and splice sites counts
• A03 - aggregated junctions with annotation status and splice site nucleotides
• A04 - aggregated junctions with selected strand and constrained splice sites
• A06 - aggregated counts filtered by annotation status and entropy
• A07 - splicing indices
• E06 - BED files with splicing indices from A06

And if --microexons is used:

• D01 - aggregated 2-splits
• D02 - aggregated constrained 2-splits
• D06 - extracted microexons from constrained 2-splits

Requirements

IPSA-nf is configured to run using the Docker container engine by default. See the included
Dockerfile for the configuration details.

In order to run the pipeline with Doecker the following dependencies have to be met:

• Java 7/8
• Nextflow 0.24.x (or higher)
• Docker 1.10 (or higher)

The pipeline can also be used without Docker by installing the following software components on your system (natively or by using Environemnt Modules):

• maptools
• sjcount
• samtools
• Perl standard modules