Question: ChIP-Seq density plot of the TSS regions?
gravatar for Susmita Mandal
18 months ago by
Susmita Mandal60 wrote:

Hello everyone,

I have done ChIP-Seq analysis alllele specific way somehow, I think. I aligned the ChIP-Seq data to both the parental genomes and then I merged the aligned BAM files and I did peak calling on the merged BAM file using MACS2. I got a BED file and a bedgraph file from it. I intersected that bedgraph file with the individual parental VCF files and got allele specific bedgraph files that converted to bigwig and I visualized in the IGV. Now I want to quantify those data and plot density plots of the TSS of the genes allele specific wise. After reading many papers and blogs and biostars, I got few words like spanning, windows, variable step and deeptools and what not. I know how to use deeptools but I don't think its giving results as I want. So can anyone make me explain step by step as how to do it. Any help is appreciated.

Thanks in advance.


ADD COMMENTlink modified 18 months ago by i.sudbery8.5k • written 18 months ago by Susmita Mandal60

I know how to use deeptools but I don't think its giving results as I want

How is that? I.e. what type of result to you get and what do you not like about it?

ADD REPLYlink written 18 months ago by Friederike6.0k
gravatar for i.sudbery
18 months ago by
Sheffield, UK
i.sudbery8.5k wrote:

The tool bam2geneprofile from cgat can produce TSS metagene plots from bigwig files.

I don't really understand how you converted a MACS2 bigwig into allele specific bigwigs, but if thats really what you've got, then after installing cgat simply run:

cgat bam2geneprofile --method=tssprofile --gtf-file=gene-models.gtf.gz --output-pattern=maternal.%s
cgat bam2geneprofile --method=tssprofile --gtf-file=gene-models.gtf.gz --output-pattern=paternal.%s

See cgat bam2geneprofile --help for ways in which profiles can be normalised.

I've also found that removing overlapping genes can make a big difference to metagene profiles. I don't know if anyone has a better way to do this, but I do it using a combination of cgat and bedtools with:

cgat gtf2gtf --method=merge-transcripts -I gene-models.gtf.gz \
                 | cgat gff2bed --is-gtf -L gene-models.filtered.log \
                 | bedtools slop -l 1250 -r 1250 \
                                 -s -i - -g contigs.tsv \
                 | sort -k1,1 -k2,2n \
                 | bedtools merge -c 4 -o count \
                 | awk '$4>1' \
                 | bedtools intersect -v -a gene-models.gtf.gz -b - \
                 | bgzip > gene-models.filtered.gtf.gz
ADD COMMENTlink written 18 months ago by i.sudbery8.5k
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