RNASeq workflow inspired from Trapnell et al, 2012

The 2012 Nature Protocol article by Trapnell C and colleagues ([PMID: 22383036]) is very well written and introduces to the field of RNASeq with a nice full workflow. You will find below text and pieces of code that are directly derived from the original workflow and propose alternatives or discussions based on it.

# group reads and run tophat

* copy all read files in a common folder
* rename each sample group with a unique prefix followed by '_reads.fq' (.gz compression is allowed)
* edit the top of 'tophat_all.sh' to reflect your reference genome and transcriptome as well as the number of threads (cpu) avaiblable on your machine.
* come to the current path in terminal
# execute 'tophat_all.sh'
 tophat_all.sh

# run each pairwise cuffdiff comparison.

* move back to where the read files are stored
* select two sample groups to be compared
* execute 'cuffdiff_2.sh' and provide information to designate the two groups
# example to compare NI and DEX groups using 16 threads
 cuffdiff_2.sh 16 "NI_" "DEX_"

# run a 4-way cuffdiff comparison.

* move back to where the read files are stored
* select two sample groups to be compared
* execute 'cuffdiff_4.sh' and provide information to designate the two groups
# example to compare NI, DEX, GW, and DG groups using 16 threads
 cuffdiff_4.sh 16 "NI_" "DEX_" "GW_" "DG"

# run all four pairwise and the 4-way comparison in one go.

* move back to where the read files are stored
* edit the bash script 'cuffdiffAll.sh' to match all sample groups and set the number of threads in it
* execute 'cuffdiffAll.sh'
 cuffdiffAll.sh

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