Bamcoverage- RPKM or 1x for normalization?

[Ying Sun]

Hi, 

I have 20 ChIP-Seq single end read bam files I am trying to generate bigwig files for so I can use them in the computeMatrix argument and then create a heatmap of how the peaks pile up around the TSS. I want to normalize my samples by the number of reads mapped to nuclear chromosomes. Which argument should I use so I can compare the 20 files I have to each other? I also have 200 bp fragments and sequenced to 75 bp so I'm guessing I would need to extend my read length. I'm also wondering if you could explain why the binSize argument is necessary and what the default is? 

Thanks! 

[Vivek Bhardwaj]

Hi

You can use the argument --normalizeTo1x and provide the genome size that you are working with. Another option is to normalize to RPKM, which doesn't require you to input genome size. After normalization (either 1x or RPKM) your files will be comparable. See the bamCoverage documentation for details of all options.

The binsize that you would choose would become the maximum resolution of the bigwig file. Default is 50 bases. You can reduce it to increase resolution.

Best Wishes

Vivek

--------

Vivek Bhardwaj

PhD Candidate | International Max Planck Research School 

Max Planck Institute of Immunobiology and Epigenetics

Stübeweg 51, Freiburg

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