Differential splicing analysis based on isoform expressions with NBSplice.

This repository has the code for generation of simulated RNA-seq datasets, NBSplice evaluation and comparison with commonly-used R packages for differential splicing detection.

Merino, G.A. & Fernández, E.A. (2019). Differential splicing analysis based on isoforms expression with NBSplice.

NBSplice is an R package able to predict isoform relative expression and its change to infer differential gene alternative splicing. Our study presents the evaluation of the developed tool using a simulated RNA-seq database. This synthetic data was generated from a real RNA-seq experiment where expression profiles were modified to controll the differential splicing patterns of genes.

The structure of this repository is as follows:

• SoftwareCodes

  • bash_scripts: Directory containing the scripts used for processing RNA-seq data
  • R_scripts: Directory having the R scripts used to perform differential splicing analysis

• Data: Simulated isoform expression matrices

Each directory contain a README and a HOWTO file.

The fastq files corresponding to the human samples can be downloaded from ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByStudy/sra/SRP/SRP002/SRP002628

• The normal samples used are:

  • SRR057649
  • SRR057650
  • SRR057651
  • SRR057652

• The tumor samples used are:

  • SRR057631
  • SRR057643
  • SRR057645
  • SRR057648

The human reference files can be donloaded from:

• Genome FASTA file, ftp://ftp.ensembl.org/pub/release-75/fasta/homo_sapiens/dna/Homo_sapiens.GRCh37.75.dna.primary_assembly.fa.gz

• Transcriptome FASTA file, ftp://ftp.ensembl.org/pub/release-75/fasta/homo_sapiens/cdna/Homo_sapiens.GRCh37.75.cdna.all.fa.gz

• Annotation GTF file, ftp://ftp.ensembl.org/pub/release-75/gtf/homo_sapiens/Homo_sapiens.GRCh37.75.gtf.gz

Installation of the following software is necessary:

• SRA toolkit (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software)

• Bowtie (http://bowtie-bio.sourceforge.net/index.shtml)

• Samtools (http://www.htslib.org/download/)

• RSEM (http://deweylab.biostat.wisc.edu/rsem/)

• STAR (https://github.com/alexdobin/STAR)

• KALLISTO (https://github.com/pachterlab/kallisto)

Installation of the following R packages is required:

• NBSplice (https://github.com/gamerino/NBSplice)
• DEXSeq (http://bioconductor.org/packages/DEXSeq/)
• DRIMSeq (https://bioconductor.org/packages/DRIMSeq/)
• RATs (https://github.com/bartongroup/RATS)
• BiocParallel (http://bioconductor.org/packages/BiocParallel/)