Hello, I am using "featureCounts" in Rsubread package for analyzing bulk RNA-seq of drosophila. Since there is no inbuilt annotations of drosophila, I am trying to use a gtf file in the homepage of iGenomes (dm6).

I made bam files using Rsubread referring to dm6, then I tried to use the following codes.

library(Rsubread)
library(limma)
library(edgeR)
targets <- readTargets("wholeflyseq.txt")
fc <- featureCounts(files=targets$OutputFile,annot.ext="genes.gtf",isGTFAnnotationFile=TRUE)

(genes.gtf is in Drosophila_melanogaster/UCSC/dm6/Annotation/Genes/genes.gtf.)

Then, there was an error message as follows.

Paired-end reads were detected in single-end read library

Actually, when I made bam files using Rsubread, there were both "properly paired" and "not properly paired" fragments. I would appreciate it if you could inform me of how to deal with this error.

Also, I am not sure if the annotation gtf file I used is appropriate or not in this case. If there is any other recommendation of the annotation files, I am grateful if you could let me informed.

Try isPairedEnd=TRUE