I am trying to view a few ATACseq and Chipseq bigwig files in UCSC. I have identified the peaks for each file, converted them to bigwig, and was able to upload them and see them in UCSC genome browser. However, what I see in full mode is a bunch of vertical lines:
I have also converted one of the bigwigs to wig, and the head shows as
#bedGraph section chr1:4768516-52764353
chr1 4768516 4768642 1
chr1 4769922 4770097 1
chr1 4780151 4780361 1
chr1 4785434 4786005 1
This is because every read is being plotted, so the Y axis goes between 0 and 1. I.e., there is no windowing happening. So here I have a few questions
- I'm wondering how I can specify the windows? In UCSC, the window is given with mean+whiskers, max, etc., so it will always show
1as the end result. But shouldn't it be computing the coverage in a given range? - Should I compute the coverage myself in a
.bedor.bedgraph, convert to.bw, and then upload? - Is the windowing done on the bigwigs, or is it done in the UCSC genomebrowser?
- Am I making any mistake or misunderstanding anything?
1 answer
I generate bedGraphs using deepTools bamCoverage for this purpose. This loop will do it, taking an input BAM file listing as input:
mkdir -p out/ ;
mkdir -p out/UCSC/ ;
cat BAM.list | sort | while read BAM ;
do
echo "Processing ""${BAM}""..." ;
echo `samtools sort -O BAM -o tmpsort.bam "${BAM}"` ;
echo `samtools index tmpsort.bam` ;
outfile=$(echo "${BAM}" | cut -f3 -d"/" | cut -f1-3 -d"_") ;
bamCoverage \
--bam tmpsort.bam \
--binSize 25 \
--smoothLength 40 \
--outFileName out/UCSC/"${outfile}".bedGraph.tmp \
--outFileFormat bedgraph \
--region chr2:49918505:51032561
echo "track type=bedGraph name=""${outfile}"" description=""${outfile}"" color=100,100,100" > out/UCSC/"${outfile}".bedGraph
cat out/UCSC/"${outfile}".bedGraph.tmp >> out/UCSC/"${outfile}".bedGraph ;
rm out/UCSC/"${outfile}".bedGraph.tmp ;
done ;
rm tmpsort.bam tmpsort.bam.bai ;
Please edit this code where necessary.
The config, after upload to UCSC, could then look something like this:
Kevin
Oh, you do not have access to the BAMs?
TagDirs - from HOMER? If 'yes', then HOMER has a function that can make UCSC bedGraph files (see HERE)
If I convert bigWig to bedGraph with bigWigToBedGraph, I still obtain a smoothed bedGraph file for UCSC. Be sure that you have the header of your bedGraph correct:
track type=bedGraph name="test" description="test" color=100,100,100
chr1 10000 10003 0.01789
chr1 10003 10101 0.03578
chr1 10101 10104 0.01789
chr1 11297 11375 0.00716
chr1 11576 11578 0.00596
chr1 11578 11604 0.01342
chr1 11604 11677 0.01789
chr1 11677 11679 0.01193
chr1 11679 11705 0.00447
chr1 11714 11732 0.00596
chr1 11732 11767 0.01044
chr1 11767 11815 0.01342
chr1 11815 11824 0.00745
chr1 11824 11833 0.01193
chr1 11833 11851 0.00745
chr1 11851 11867 0.01491
chr1 11867 11868 0.01193
chr1 11868 11876 0.0164
chr1 11876 11917 0.02714
chr1 11917 11925 0.03012
chr1 11925 11952 0.02564
If all else fails, then, indeed, just summarise the coverage yourself using the information in the bedGraph that you have already created.
Dear Kevin, thanks so much, this solved my problem! I found a way to get the bedgraphs and it worked.
I would just like to ask one final question: in general, when you are computing coverage for visualization in a UCSC, or IGV, what window sizes do you usually use? I understand this is very much case dependent, but I'm just trying to get a general sense of what would a sensible windowsize be? 15bps? 25bps?
Oh, can be anything, really. A smaller window size will produce a larger file, but provides greater resolution. I mean, if you're going to be zooming in on individual regions, it may help to have a greater resolution (small window size) so that it doesn't look 'blocky'. If you're going to just look at entire gene bodies, then perhaps you can use a larger window size.
Given the data that you have, you probably do not have much room for altering the window size.
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Thanks for your comment. I may be misunderstanding what you want to say, but I have no problems uploading my data to UCSC. The screenshot I gave is from there actually. I have also updated my question's title as the focus is on the "windows" part