I am trying to analyze my spiked-in ChIP-Seq data by scaling my original aligned BAM files. My workflow is as following,
Use ChIPSeqSpike (https://www.bioconductor.org/packages/release/bioc/html/ChIPSeqSpike.html) to get scaling fact for exogenous spiked-in drosophila chromatin across different samples.
Use bamCoverage to get bigwig files by including the arguments: --scaleFactor 1.092883 -bs 10 --normalizeUsing RPGC --effectiveGenomeSize 2685511504 --extendReads 150
For the input seq data, only use the RPGC scaling in bamCoverage to get bigwig files
Convert individual bigwig files to wig using bigWigToWig (ucsc), then wig2bed (BEDOPS) to get bed files
I already used MACS2 to call peaks using the original BAM files and obtained the merged peaks across my samples in comparison. The purpose to obtain the scaled bigwig files is to perform the differential binding analysis across different samples, since I concerned the ChIP variance due to the altered ChIPed proteins across my samples in comparison.
But now I don't know how to further perform the differential peaks analysis. I know DiffBind requires original BAM files, which I don't know how to produce the scaled BAM files, other than the scaled bw/bedgraph/bed files I obtained above. I also had problems using the scaled bed files to build the TagDirectory in HOMER (failed in fragment length estimation), which can do such analysis using getDifferentialPeaks command.
I will really appreciate if someone can guide me to solve my problems. Thank you!
Xiaoyong Fu Baylor College of Medicine