Im having difficulty while running the APAlyzer software package on paired-end reads. Has anyone used this software before?
It looks like APAlyzer hardcodes singleEnd = TRUE in its internal call to featureCounts (see line 37 of APAlyzer.R in the source code). This means that it is currently set up to treat all input BAM files as single-end, regardless of whether your reads are paired-end.
There is an open issue about this on GitHub dating back to 2022: How to run PASEXP_3UTR with paired end reads? - unfortunately, it remains unresolved with no response from the maintainers.
As a workaround, you could fork the repository and modify the code yourself to set singleEnd = FALSE (or make it a user-configurable parameter), then reinstall the package locally via devtools::install_github() or similar. Here's a quick example of what the modified line might look like in APAlyzer.R:
count <- featureCounts(files = flS, annot.ext = UTRdb, isGTFAnnotationFile = FALSE, nthreads = nthreads, isPairedEnd = TRUE, strandSpecific = StrandtypeN, primaryOnly = TRUE, countMultiMappingReads = FALSE)
Be sure to test this carefully, as changing it could affect how overlaps are handled for paired reads.
If that's not feasible, you may want to reach out to the authors directly via the GitHub issue or email (check the Bioconductor page for contact info). In the meantime, please share the exact error message you're seeing and your code / command - that may help pinpoint if there's another issue at play.
Have you confirmed that your BAM files are properly sorted and indexed? Also, the vignette has some examples for basic usage: APAlyzer Vignette
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That is not enough information for anyone to help. Please describe what kind of difficulty. Have you checked program vignette, if one is available.