Expression analysis

Installing rsem

We’ll be using the RSEM package to do some expression analysis. To install it:

cd /root

curl -O http://deweylab.biostat.wisc.edu/rsem/src/rsem-1.2.5.tar.gz

tar xzvf rsem-1.2.5.tar.gz
cd rsem-1.2.5
make

And now add this directory into your PATH, which is where Unix looks for things to run:

echo 'export PATH=$PATH:/root/rsem-1.2.5' >> /root/.bashrc
source /root/.bashrc

Installing bowtie

If you didn’t install bowtie on this machine already (e.g. as part of Doing a small de novo mRNAseq assembly with Trinity), RSEM needs it; do:

cd /root
curl -O -L http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip
unzip bowtie-0.12.7-linux-x86_64.zip
cd bowtie-0.12.7
cp bowtie bowtie-build bowtie-inspect /usr/local/bin

Running rsem

Go to a working directory on /mnt:

cd /mnt
mkdir rsem
cd rsem

Now, grab the reference transcriptome:

curl https://s3.amazonaws.com/public.ged.msu.edu/coral-mini-assembly.fa.gz |      gunzip > coral.fa

and some reads:

curl -O http://athyra.idyll.org/~t/larva-ct-800k.fq.gz
curl -O http://athyra.idyll.org/~t/settled-ct-800k.fq.gz

You’ll need to uncompress the reads:

gunzip *.gz

Now, prepare the reference for RSEM:

rsem-prepare-reference coral.fa coral

Here, ‘coral.fa’ can be one of several files (comma-separated) in case you have multiple different reference data sets. In this case it’s the transcriptome you generated, but there are lots of options (see the docs for rsem-prepare-reference).

And, finally, calculate the expression levels for the ‘settled’ and ‘larva’ data sets:

rsem-calculate-expression settled-ct-800k.fq coral settled
rsem-calculate-expression larva-ct-800k.fq coral larva

(See the rsem-calculate-expression docs for more info.)

This outputs a couple files; the two most interesting, for now, are ‘larva.genes.results’ and ‘settled.genes.results’. These contain the following data:

head larva.genes.results

should show you

gene_id transcript_id(s)        length  effective_length        expected_count TPM      FPKM
comp0_c0_seq1   comp0_c0_seq1   291.00  291.00  3.00    102.12  164.47
...

If you’ve installed Dropbox (see ../tutorials-2013/installing-dropbox), you can now copy the files to Dropbox:

cp larva.genes.results settled.genes.results ~/Dropbox/

and you can open them directly in Excel as tab-delimited files.


LICENSE: This documentation and all textual/graphic site content is licensed under the Creative Commons - 0 License (CC0) -- fork @ github.
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