After mapping of the sequencing reads to the human genome, bam files were converted to bed using bedtools’ (v2.29.2) “bamtobed” and only sequence chromosome and start information retained. Optional: split files by chromosome for better handling. (example files: chr1.input_start.bed, chr1.chip_start.bed)
Define bin size (e.g.: 10kb, chromosome size information necessary) and count the number of reads within each bin (function: 01_countReadFrequency_example.R). Optional: Index files to remove bins without reads later on.
For normalization by input, keep bins > 0 in input. Match bins in chip files and divide ChIP read frequency by input read frequency. Apply normalization by mean ChIP read frequency (function: 02_inpDiv_normByMean.R)
The resulting files contain the index of the bin and the normalized read frequencies.
Upload files:
hg19_chrSizes.bed
chr1.input_start.bed
chr1.chip_start.bed
01_countReadFrequency_example.R
02_inpDiv_normByMean.R
Copyright: Content may be subjected to copyright.
How to cite:
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
Kirstein, N, Hyrien, O, Audit, B and Schepers, A(2021). Binning approach and normalization. Bio-protocol Preprint. bio-protocol.org/prep1072.
Kirstein, N., Buschle, A., Wu, X., Krebs, S., Blum, H., Kremmer, E., Vorberg, I. M., Hammerschmidt, W., Lacroix, L., Hyrien, O., Audit, B. and Schepers, A.(2021). Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones. eLife. DOI: 10.7554/eLife.62161
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