An Example Differential Expression Analysis Using RNA-Seq Data

This is an example analysis of RNA-seq data using open source tools R and Bioconductor. It starts with raw reads downloaded from the Short Read Archive (SRA), does quality assessment and improvement, mapping, and analysis.

Data

The data for this comes from Zhang et al., 2011, Genome-wide mapping of the HY5-mediated genenetworks in Arabidopsis that involve both transcriptional and post-transcriptional regulation (http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2010.04426.x/full). It is composed of two Arabidopsis thaliana ecotype Col-0 sets, one hy5 mutant and one wild type. The SRA accession is SRX029582.

• GEO:GSM613465: wild type samples
  • SRR070570: replicate 1
  • SRR070571: replicate 2
• GEO:GSM613466: hy5 mutant samples
  • SRR070572: replicate 1
  • SRR070573: replicate 2

These were sequenced on an Illumina Genome Analyzer after oligo(dT) selection and random hexamer priming.

Downloading

Read data was acquired from the SRA via wget, and checked with:

cd data/raw-reads/
md5sum -c *sra.md5

The TAIR10 Arabidopsis Genome was downloaded with:

wget -nd ftp://ftp.arabidopsis.org/home/tair/Sequences/whole_chromosomes/*fas

-nd tells wget not to mirror the directory structure.

FASTQ Extraction

FASTQ data was extracted with NCBI's SRA Toolkit's fastq-dump, used with xargs and find to run this in parallel:

find . -name "*sra" | xargs -n1 -P4 /share/apps/sratoolkit/fastq-dump

Process a Genome for GSNAP

cd data/athaliana-genome
gmap_build -d athaliana10 -C *.fas

The -C option tries to extract chromosome information from the FASTA header.

Raw Sequence Quality Assessment

First, we assess the quality with qrqc; see raw-read-qa.Rmd.

Scythe

sycthe is a 3'-end quality trimmer. Illumina adapters are removed from the raw reads:

find data/raw-reads/ -name "*.fastq" | xargs -n1 -I{} basename {} .fastq | xargs -n1 -P4 -I{} /share/apps/scythe/scythe -q sanger -a /share/apps/scythe/solexa_adapters.JNF.fa -o data/improved-reads/{}-trimmed.fastq data/raw-reads/{}.fastq

Sickle

sickle is a tool for trimming low-quality bases off of the 5'-end and 3'-end of reads.

Aligning Reads with GSNAP

I use a script (map-gsnap.sh) to process each alignment file. The purpose of using the script is that I can use it with xargs. Because gsnap returns results to standard out, we need to wrap the call in a script to allow this to be parallelizable.

find data/improved-reads/ -name "*final.fastq" | xargs -n1 -P4 bash map-gsnap.sh

I am using -N1 with gsnap, which allows for novel splicing. gsnap also allows for known splicing junctions to be used.

Converting SAM files to BAM files

Another task for xargs. As before, I use basename to keep just the unique file name as a key, and run samtools with the correct directory and extension. This allows me to change the extension too.

find data/alignments/ -name "*sam" | xargs -n1 -I{} basename {} .sam | xargs -n1 -I{} -P4 samtools view -b -S -o data/alignments/{}.bam data/alignments/{}.sam