In order to get start analysing my CAGE library using CAGEfightR I prepared the required BIGWIG files as fellow: End-to-end bowtie2 alignment against UCSC.mm10, SAM to sorted BAM files and made the BIGWIG files using CAGEr getCTSS() function and exportCTSStoBedGraph(format = "BigWig”).

For quantify CTSSs, the manual example used BSgenome.Mmusculus.UCSC.mm9 as a genome seqinfo. Since I already aligned with mm10 I changed the example and run it like this:

CTSSs <- quantifyCTSSs(plusStrand = bwplus, minusStrand = bwminus, genome = seqinfo(BSgenome.Mmusculus.UCSC.mm10), design = design_matrix)

Well, the immediate unidirectional clustering using quickTSSs(CTSSs) resulted with an error:

Error in stopifwronglength("'seqnames'", anslen) : 'seqnames' must have the length of the object to construct (1) or length 1

So I tried the mm9 as suggested:

CTSSs <- quantifyCTSSs(plusStrand = bwplus, minusStrand = bwminus, genome = seqinfo(BSgenome.Mmusculus.UCSC.mm9), design = design_matrix)

and got the error:

Error in mergeNamedAtomicVectors(seqlengths(x), seqlengths(y), what = c("sequence", : sequences chr1, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chrX, chrY have incompatible seqlengths: - in 'x': 195471971, 130694993, 122082543, 120129022, 120421639, 124902244, 104043685, 98207768, 94987271, 90702639, 61431566, 182113224, 160039680, 156508116, 151834684, 149736546, 145441459, 129401213, 124595110, 171031299, 91744698 - in 'y': 197195432, 129993255, 121843856, 121257530, 120284312, 125194864, 103494974, 98319150, 95272651, 90772031, 61342430, 181748087, 159599783, 155630120, 152537259, 149517037, 152524553, 131738871, 124076172, 166650296, 15902555

Do I need to re-align my library against the mm9 and make again the BIGWIG files? I'm quit confused since later the manual uses the mm10 for promoter calling. Thanks, Ariel