Hi there! I have no bioinformatics background. Just started learning RNA sequencing from tutorials. So prior apologies for asking any basic questions. Today I have 2: A) How do I produce coverage plots like the ones in the attached figures? I have tried IGV but is there any way to do it via UCSC Genome Browser? I need the sense and antisense strand coverage in the same plot preferably.
B) Coverage plots are created from .BAM files. If I am comparing a WT and a Mutant sample by read coverage plots, how does it make sense if the exact same number of reads didn't end up in both samples? Increased number of Reads in the Mutant will automatically lead to greater read coverage of any particular feature right? Then how does it make sense to show read coverage without having proper controls? Many publications have such plots so obviously I am missing something. Or not understanding something basic?
A: You could use UCSC browser but you would need to find a place to host your BAM files on web so they can be projected on UCSC browser. Why don't you use IGV (and use right click options to sorts reads based on strand, use squished mode to compress the read representation).
B: You can't visually compare any two regions. Two samples may have had very different number of reads to begin with. You could use deepTools bamcoverage plots to show normalized data.
I have been using IGV. I can sort and modify the alignment track. However the presentation is not as clean. And I can't seem to do anything to the coverage track. No sorting, modification anything. Can't even make the data range label larger.
Could you tell me the option on UCSC genome browser? Can't seem to find it. I am using galaxy so no issues with the hosting of BAM files.
For B, I'll definitely check that and get back if I have any issues. Thank you so much for responding and suggesting the tool.