Pipeline for analyzing standard Illumina RNA-seq data

The pipeline follows the steps outlined below to analyze standard Illumina RNA-seq data:

1. Pre-Alignment QC
2. Alignment
3. Provision for visualizing alignments using IGV
4. Alignment QC
5. Expression and Differential Expression Analysis
6. Reference-Free Abundance Estimation using Kallisto
7. Isoform Discovery and Alternative Expression a) Reference Guided Transcript Assembly b) Differential Splicing c) Splicing Visualization

##1) Pre-Alignment QC Run FASTQC on RNA-seq reads to assess quality of data If data are in folder $RNA_seq/data

cd $RNA_seq/data
fastqc *.fastq.gz

Alternatively, use http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ to generate QC reports for each set of read data

2) Alignment

#Create directory for alignment data
mkdir alignments
cd alignments
for f in `cat ${files}`; do STAR --genomeDir ${GENOMEDIR} \
--readFilesIn fastq/$f\_R1.fastq fastq/$f\_R2.fastq \
--runThreadN 24 --outSAMtype BAM SortedByCoordinate --outFileNamePrefix aligned/$f.; done

3)