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We are using featureCounts for both and single-end and pair-end data. In the command we always have the '-p' flag on, assuming it will be ignored for single-end data. However, we notice that the function of '-M' is affected because of '-p' flag.
When we run single-end data without the -M flag, all the reads with NH > 1 are ignored, as expected. In the test data attached, we get a read count of 4. With -M flag, we get a count of 16, including reads with NH > 1, again, as expected.
However, when we run single-end data without the -M flag but with '-p' flag, the multi-mapping reads (NH > 1) are no longer ignored and we get a read count of 16.
The '-p' should have no effect on filtering multi-mapping reads. I'm attaching a sample sam and gtf file where this behavior may be replicated.
When we run single-end data without the -M flag, all the reads with NH > 1 are ignored, as expected. In the test data attached, we get a read count of 4. With -M flag, we get a count of 16, including reads with NH > 1, again, as expected.
However, when we run single-end data without the -M flag but with '-p' flag, the multi-mapping reads (NH > 1) are no longer ignored and we get a read count of 16.
The '-p' should have no effect on filtering multi-mapping reads. I'm attaching a sample sam and gtf file where this behavior may be replicated.
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