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Reads detected/"spill over" on introns after using bamCoverage function from deepTools

Hello! We recently used the recent version of bamCoverage from the deepTools suite for our ribo-seq and RNA-seq bam files to generate bigwig files. The conversions ran smoothly, however when we loaded the bigwig files into IGV, we noticed some reads spilling over to some exon-intron junctions when we zoomed into each track. We believe these are not genome misannotations because the non-normalized/non-bigwig bam files don't show these junction reads. Any advice is appreciated. Thank you!

Best, Kat

bamcoverage deeptools bigwig intron
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updated by cpad0112 cpad0112 • written by kdcastillo • kdcastillo

Hello, it would help if you add code and screenshots to understand the problem. My first guess when it comes to local anomalies is the chosen bin size and extension parameter (-bs -e).

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written by ATpoint ATpoint

enter image description hereHello! I agree that it might be due to bin size, I tried the default and bin size of 10, and I am still getting the same issue. But I attached the script (for bin size 10) and the example image of the problem I'm seeing.

bamCoverage -b filename.bam -o filename.bin10rpkm.bw --normalizeUsing RPKM -bs 10

Thanks for the response!

Kat

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updated by i.sudbery i.sudbery • written by kdcastillo • kdcastillo

Is this riboseq data? can you check the length of the retained bases? If it is riboseq, you may have to set offset.

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written by cpad0112 cpad0112

Did changing the bin size end up fixing this issue for you?

Franco

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written by FCarranz • FCarranz

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