Mapped Reads Into A Gene Level Count

Question

[joaslucas]

Hi everyone, This is a couple of simple questions

After mapping reads (RNA-seq) against a reference bacterial genome I need to know how many reads I got for each gene. How do I do that, which program should I use? And finally, big genes will have much more reads than smaller genes, and that cannot be accounted for DE, Is this problem solved in the normalization step?

Thanks, I am a biologist tying to understand RNA-seq. I ve read some papers but I still far from understanding the whole process.

Thanks for the help.

Thanks.

Answer 1

[swbarnes2]

A simple strategy:

Make a .bed of genes coordinates

Use BEDTools to find out how many reads hit each gene

Correct for gene length

Correct for differing # of reads between samples

[zizigolu]

Sorry, from where can I get the gene coordinates? I have a bam file then how I can have a gene coordinates?

Answer 2

[macmath]

Check these links:

• http://bioinformatics.oxfordjournals.org/content/early/2013/01/16/bioinformatics.btt016.abstract
• http://www.rna-seqblog.com/tag/data-analysis-pipeline/

Answer 3

[Asaf]

Another great place to start is in this Galaxy exercise: https://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise Since you're dealing with bacteria you should probably use Bowtie and not Tophat but I think it won't have any effect.