Hi Everyone,
I have 2 bam files which I produced by mapping the reads with bowtie using hg19. This is a CHIP seq data for one of the histone marks, where one file is input(control) and other file is the chip sample. I want to show for certain genes which I am interested in, read plots around the Transcription Start Sites(TSS). Let's say I have 6 genes of interest, so in one TSS plot(for that histone mark) I want to see how histone marks are represented for different genes.
How can I do it?
Thanks
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written
by
Varun Gupta
5 answers
I've had great success following the BEDTools protocol for plotting occupancy. Starts on page 18.
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written
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James Ashmore
I guess you can find that protocol in the following URL.
https://github.com/arq5x/lab_website/blob/master/pdf/bedtools.protocols.pdf
The same can be achieved using deepTools.
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written
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Arup Ghosh
See if this Software for drawing peak shapes or this Visualizing average read coverage across features with varying length thread helps.
DeepTools will do exactly what you need quickly and nicely. You can use our Galaxy instance or install deepTools using python pip command (see our github page for that).
First you need to transform you bam file to a bigwig file (using the tool called bamCoverage from deepTools). The following code will probably work:
bamCoverage -b file.bam -o coverage.bw
# compute the coverage for a region 3000 bp downstream and upstream of the TSS
computeMatrix reference-point -R my_genes.bed -S coverage.bw \
-o gene_cov --afterRegionStartLength 3000 \
--beforeRegionStartLength 3000 --referencePoint TSS
heatmapper -m gene.cov -o heatmap_and_profile.pdf
profiler -m gene.cov -o profile.pdf
As a recommendation, I suggest to use a log2 ratio of ChIP-seq / input instead of the coverage as this can be biased.
HI Fidel,
I was trying to use Deeptools, but don't you think for the chip sample, I should use bamCompare and normalize and get a normalized bigwig file
Something like this
bamCompare --bamfile1 chip1.bam --bamfile2 Input.bam --ratio log2 --scaleFactorsMethod SES --binSize 50 -o chip_vs_input.bw
And then should I use computeMatrix command? I think we should always normalize the chip with input(as you say log2 normalization)
Also what does my_genes.bed file consists of?
I think my bed files should have the TSS site of the gense I am interested in : something like this:
chr1 34565 34566 gene_b 100 +
chr17 43300230 43300231 gene_a 100 -
Will the heatmap show both these genes for the particular histone mark I am interested? I might increase the list of genes in my bed file
Let me know
Thanks for the help
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written
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Varun Gupta
Yes, use bamCompare. Be careful with the scale factor. The SES method is good for sharp peaks but not for broad marks, so choose based on the mark that you have.
To get the TSS correctly the only requirement is that the bed file contains the strand information, you don't need to specify the TSS. The usual bed for genes like from UCSC is what is needed. The heatmap will show the two genes but I think it works best when you have many more genes.
Hi Fidel,
Thanks for the explanation. The default scale method is SES and it is good for sharp peaks as you say but, for peaks with H3K27ac , those are usually broader, what scale factor should I use?
About the bed file: What information should I include in it? The one I am using above, is it wrong??
I recommend you to take a look at the bamCompare manual to understand the scaling methods. The bed file that you are using will also work.
Hi Fidel,
I am trying to view a heatmap for around 70 of my genes at TSS. Is there a way I can label those genes on the heatmap. All it labels is genes. Also is there a way to label them horizontally and not vertically? That would be a great help. Thanks for the wonderful tool.
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written
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Varun Gupta
Also NGS-Plot is a nice tool!
I want to add Genomation and my post here
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written
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Ming Tommy Tang
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