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Mapped Reads Into A Gene Level Count

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.

rna-seq counts
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updated by Ram Ram • written by joaslucas • joaslucas

3 answers

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

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written by swbarnes2 swbarnes2

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

• 1 views • link
updated by Ram Ram • written by zizigolu zizigolu

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.

• 0 views • link
updated by Ram Ram • written by Asaf Asaf

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