Hi all,
I recently work on several ChIP-seq data sets. My traditional workflow is .fastq/.fastq.gz -> .sam (via Bowtie) -> MACS (after BAM conversion) -> wig to bigwig conversion. Thus, my usual output are .bw track files that I then view through UCSC genome browser, with the y axis (track height) scaled to sequencing depth (for example the Bowtie alignment reported 15M aligned reads, y axis would be 1 - 15).
However, recently I encountered an issue with MACS, or more precisely, wigToBigWig tool:
Overlap on chr4 between items starting at 29999999 and 30000000. Please remove overlaps and try again Fatal error: Matched on Error Error running wigToBigWig.
I followed the suggestion of the Galaxy team and tested out MACS 2 and succeeded constructing a BedGraph file, which I then converted to .bw track to view through UCSC genome browser as usual. The result was very good. However, I noticed that the y-axis scaling is different from what I usually have with MACS -> bigwig method. In MACS2 -> bigwig case the peaks are only visible with y-axis range being 0.4 - 0.6, while in traditional MACS -> bigwig case peaks are usually visible with the range being 1 - 15 (as I mentioned above). Is there a way to change the scaling of the track created by MACS2 -> bigwig method to match the scaling scheme that I usually use for with MACS -> bigwig?
Here is the link to the history in question: https://usegalaxy.org/u/sushivision/h/tks367b3-0113
I must admit that my understanding of ChIP-seq analysis is very shallow. Please feel free to critique my practices. I would greatly appreciate an explanation for the meaning of track height if anyone can provide it.
Best,
Bao
Track visualization can be customized at UCSC (see their help docs, or just click into the track details and adjust).
Conversion using wigToBigWig is not the source of the score/depth difference - the underlying statistics come from MACS/2 output. My initial guess is that there is a difference in how the parameters used with the MACS vs MACS2 runs were applied that impacted scores/scaling. There are other differences between the two tool versions that will make any rerun job using the newer tool version produce slightly different results, even when compatible parameters are used.
I'll take a look at the new runs (and others can as well!). Thanks for testing out this suggested solution to the prior problem. More feedback after review. - Jen
I have tried to run MACS and MACS2 on the same alignment result for several data sets. Surprisingly the results I received are very similar. In some cases MACS2 yielded a higher level of noise/background. It seems that the data that I shared above is the only unique case with tiny peaks and clean background.
I have also found this: https://www.genomatix.de/online_help/help_regionminer/macs.html
According to the website the scoring schemes for MACS and MACS2 are the same (except quality score, but I do not think that this affect the height of peaks).
Thank you for your help! If you have found something of note please let me know.