Bamcoverage tool normalizing for sequencing depth and internal spike-in scaling factor at the same time
Daniel Bsteh
Hi,
I have a ChIPseq experiment where I have an internal spike-in control of drosophila chromatin. I did the regular analysis including alignment to both genomes (mouse and drosophila) and filtering for mappin quality, unique alignment and duplicates. So now I have filtered bam files of my ChIPseq samples with varying sequencing depth and each has their corresponding read number of drosophila spike-in control chromotin.
I can calculate scaling factors to normalize for the differences in spike-in reads between samples.
My question is now do I use these scaling factors alone by themselves or can I use them in combination with e.g --normalizeUsing RPKM to account for sequencing depth differences as well or is that not possible/necessary/advisable?
Thanks for any help you might be able to provide!
Daniel
Devon Ryan
When making the bigWig files you can supply the scaling factors you
computed with the `--scaleFactor` option. You can use this in
conjunction with `--normalizeUsing RPKM` if you like, though I
personally prefer to omit the `--normalizeUsing` option in cases like
this (to each their own).
Devon
--
Devon Ryan, Ph.D.
Email: dpr...@dpryan.com
Data Manager/Bioinformatician
Max Planck Institute of Immunobiology and Epigenetics
Stübeweg 51
79108 Freiburg
Germany
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