Looking to get some feedback on a probability weight function (PWF) curve I've generated using GOseq. As I understand it, as long as the curves fit the data points reasonably then the downstream analysis will be ok however mine look a bit strange compared to examples I've seen elsewhere. This seems to be mainly due to a few extreme outliers and I'm wondering whether these will be affecting the results unduly and whether there is a way to account for these within the nullp() function. Also whether I've made some mistake in the gene length determination... Here is my process
A) Get length data for Hg38 from Ensembl and filter for only my expressed genes (filtered by all genes with count >1 in treatment of interest - too low?):
txdb <- makeTxDbFromEnsembl("Homo sapiens", release = 89) txsByGene=transcriptsBy(txdb,"gene") lengthData=median(width(txsByGene) lengthfilt <- lengthData[names(lengthData) %in% names(gene_list)]
B) Use nullp() to generate a PWF for the expressed genes based on their respective lengths
pwf = nullp(clustVsAll, "hg38", "ensGene", bias.data = lengthfilt)
Any help/feedback much appreciated!