RNASeq Transcript Mapping and Counting (BWA and HtSeq Flavor)

The goal of this tutorial is to show you one of the ways to map RNASeq reads to a transcriptome and to produce a file with counts of mapped reads for each gene.

We will be using BWA for the mapping (previously used in the variant calling example) and HtSeq for the counting.

Booting an Amazon AMI

Start up an Amazon computer (m1.large or m1.xlarge) using AMI ami-7607d01e (see Start up an EC2 instance and amazon/starting-up-a-custom-ami).

Go back to the Amazon Console. Now select “snapshots” from the left had column. Changed “Owned by me” drop down to “All Snapshots”. Search for “snap-028418ad” - (This is a snapshot with our test RNASeq Drosophila data from Chris) The description should be “Drosophila RNA-seq data”. Under “Actions” select “Create Volume”, then ok.

Now on the left select “Volumes”. You should see an “in-use” volume - this is for your running instance, as well as an “available” volume - this is the one you just created from the snapshot from Chris and should have the snap-028418ad label. Select the available volume and from the drop down select “Attach Volume”. The white box pop up will appear - select in the empty instance box, your running instance should appear as an option. Select it. For the device, enter /dev/sdf. Now attach.

Log in with Windows or from Mac OS X.

Updating the operating system

Become root

sudo bash

Copy and paste the following two commands

apt-get update
apt-get -y install screen git curl gcc make g++ python-dev unzip \
        default-jre pkg-config libncurses5-dev r-base-core \
        r-cran-gplots python-matplotlib sysstat

to update the computer with all the bundled software you’ll need.

Mount the data volume. (This is for Chris’s data that we added as a snapshot).

cd /root
mkdir /mnt/ebs
mount /dev/xvdf /mnt/ebs

Install software

First, we need to install the BWA aligner:

cd /root
wget -O bwa-0.7.10.tar.bz2 http://sourceforge.net/projects/bio-bwa/files/bwa-0.7.10.tar.bz2/download

tar xvfj bwa-0.7.10.tar.bz2
cd bwa-0.7.10

cp bwa /usr/local/bin

We also need a new version of samtools:

cd /root
curl -O -L http://sourceforge.net/projects/samtools/files/samtools/0.1.19/samtools-0.1.19.tar.bz2
tar xvfj samtools-0.1.19.tar.bz2
cd samtools-0.1.19
cp samtools /usr/local/bin
cp bcftools/bcftools /usr/local/bin
cd misc/
cp *.pl maq2sam-long maq2sam-short md5fa md5sum-lite wgsim /usr/local/bin/

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