I have human total RNA-seq data (PE, Next-Seq) from a pilot study with two samples and am getting results from featureCounts that do not make any sense to me. I process the data as follows: (1) I perform adapter trimming using ea-utils mcf and mild quality trimming using btrim. I use STAR for alignment to the genome. STAR tells me that the first sample has 99,018,190 input reads (these are read pairs) and the 2nd sample has 126,164,150 input reads. For the first sample, 86,571,963 reads were aligned (70,579,007 uniquely), and for the 2nd sample, 112,525,928 reads were aligned (89,730,382 uniquely). Now when I run featureCounts on these data, it prints out this information:
First sample: Total fragments: 123128857, Successfully assigned fragments : 63971619 (52.0%)
2nd sample: Total fragments: 168328570, Successfully assigned fragments : 83223103 (49.4%)
and this warning:
WARNING: reads from the same pair were found not adjacent to each other in the input (due to read sorting by location or reporting of multi-mapping read pairs).
It seems that the total fragments from featureCounts do not match up at all with the read counts from STAR.