featureCounts exon-level, problems with the counting verification

[gcR]

Hey, I was asked to do some analysis at exon level with featureCounts from a sample of stem cells induced to adypocites, and this was my result:

 fc5 <- featureCounts(filevar, annot.ext = "/home/sysadm/tomics/data3", isGTFAnnotationFile = TRUE, GTF.featureType = "exon", GTF.attrType = "exon_id", useMetaFeatures = FALSE, isPairedEnd = FALSE)

//========================== featureCounts setting ===========================\\
||                                                                            ||
||             Input files : 8 BAM files                                      ||
||                           S /home/sysadm/tomics/ADI/4_GCCAAT_L001_R1_0 ... ||
||                           S /home/sysadm/tomics/ADI/8_AGTTCC_L001_R1_0 ... ||
||                           S /home/sysadm/tomics/ADI/16_GCCAAT_L002_R1_ ... ||
||                           S /home/sysadm/tomics/ADI/20_AGTTCC_L003_R1_ ... ||
||                           S /home/sysadm/tomics/ADI/28_GCCAAT_L004_R1_ ... ||
||                           S /home/sysadm/tomics/ADI/32_AGTTCC_L004_R1_ ... ||
||                           S /home/sysadm/tomics/ADI/40_GCCAAT_L005_R1_ ... ||
||                           S /home/sysadm/tomics/ADI/44_AGTTCC_L006_R1_ ... ||
||                                                                            ||
||             Output file : ./.Rsubread_featureCounts_pid3469                ||
||             Annotations : /home/sysadm/tomics/data3 (GTF)                  ||
||                                                                            ||
||                 Threads : 1                                                ||
||                   Level : feature level                                    ||
||              Paired-end : no                                               ||
||         Strand specific : no                                               ||
||      Multimapping reads : not counted                                      ||
|| Multi-overlapping reads : not counted                                      ||
||                                                                            ||
\\===================== http://subread.sourceforge.net/ ======================//

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file /home/sysadm/tomics/data3 ...                         ||
||    Features : 1193694                                                      ||
||    Meta-features : 680541                                                  ||
||    Chromosomes : 25                                                        ||
||                                                                            ||
|| Process BAM file /home/sysadm/tomics/ADI/4_GCCAAT_L001_R1_001.fastq.gz ... ||
||    Single-end reads are included.                                          ||
||    Assign reads to features...                                             ||
||    Total reads : 19649163                                                  ||
||    Successfully assigned reads : 3246600 (16.5%)                           ||
||    Running time : 0.59 minutes                                             ||
||                                                                            ||
|| Process BAM file /home/sysadm/tomics/ADI/8_AGTTCC_L001_R1_001.fastq.gz ... ||
||    Single-end reads are included.                                          ||
||    Assign reads to features...                                             ||
||    Total reads : 17658467                                                  ||
||    Successfully assigned reads : 2941765 (16.7%)                           ||
||    Running time : 0.53 minutes                                             ||
||                                                                            ||
|| Process BAM file /home/sysadm/tomics/ADI/16_GCCAAT_L002_R1_001.fastq.g ... ||
||    Single-end reads are included.                                          ||
||    Assign reads to features...                                             ||
||    Total reads : 18461466                                                  ||
||    Successfully assigned reads : 4610192 (25.0%)                           ||
||    Running time : 0.64 minutes                                             ||
||                                                                            ||
|| Process BAM file /home/sysadm/tomics/ADI/20_AGTTCC_L003_R1_001.fastq.g ... ||
||    Single-end reads are included.                                          ||
||    Assign reads to features...                                             ||
||    Total reads : 18476485                                                  ||
||    Successfully assigned reads : 4799166 (26.0%)                           ||
||    Running time : 0.61 minutes                                             ||
||                                                                            ||
|| Process BAM file /home/sysadm/tomics/ADI/28_GCCAAT_L004_R1_001.fastq.g ... ||
||    Single-end reads are included.                                          ||
||    Assign reads to features...                                             ||
||    Total reads : 16438703                                                  ||
||    Successfully assigned reads : 5479529 (33.3%)                           ||
||    Running time : 0.53 minutes                                             ||
||                                                                            ||
|| Process BAM file /home/sysadm/tomics/ADI/32_AGTTCC_L004_R1_001.fastq.g ... ||
||    Single-end reads are included.                                          ||
||    Assign reads to features...                                             ||
||    Total reads : 32599890                                                  ||
||    Successfully assigned reads : 7140596 (21.9%)                           ||
||    Running time : 1.11 minutes                                             ||
||                                                                            ||
|| Process BAM file /home/sysadm/tomics/ADI/40_GCCAAT_L005_R1_001.fastq.g ... ||
||    Single-end reads are included.                                          ||
||    Assign reads to features...                                             ||
||    Total reads : 16299496                                                  ||
||    Successfully assigned reads : 4049332 (24.8%)                           ||
||    Running time : 0.53 minutes                                             ||
||                                                                            ||
|| Process BAM file /home/sysadm/tomics/ADI/44_AGTTCC_L006_R1_001.fastq.g ... ||
||    Single-end reads are included.                                          ||
||    Assign reads to features...                                             ||
||    Total reads : 25469357                                                  ||
||    Successfully assigned reads : 6164219 (24.2%)                           ||
||    Running time : 0.79 minutes                                             ||
||                                                                            ||
||                         Read assignment finished.                          ||
||                                                                            ||
\\===================== http://subread.sourceforge.net/ ======================//

As you can see, my assigned reads vary from 15-30%, so I was asked to check why so many reads felt outside the exons (now things are getting harder for me ).

For that task I was told to use IGV, which helps me visualizing the reads compared to the genome, but I hardly understand it.

What would you do in my case? I will make a list with the assigned reads to exons, get the gene ID for those exons, and compare it using IGV, but Im not really sure if this will give me answers.

Thanks in advance, and sry for ambiguity, Im just starting

[GenoMax]

Are you sure these samples are non-stranded? What happens if you do the counting as stranded?

There is a nice user guide for IGV that describes all of its features. Carefully look in IGV (choose correct genome build!) to see if reads are mapping outside exons? What read deapth you see across the sample(s)?