How are multimapping reads counted?

[rbeagrie]

My group is currently using DropEst to process some single-cell data from erythroid differentiation, so two of our key marker genes are alpha and beta globin. Both genes are present in two identical, duplicate copies, so reads that map to these genes are output twice in our alignment bam, and one of the two alignments is randomly marked as the primary alignment. However, the globin genes are missing from the count matrix files after running dropEst - so I'm guessing multimapped reads are getting discarded? Is there a way to get DropEst to count all the primary alignments (and primary alignments only) regardless of whether the read is uniquely mapped?

[rbeagrie]

I edited the bam files to keep only primary alignments and rewrite "NH:i:2" to "NH:i:1" so that dropEst does not know they are multimapped. However this doesn't fix the problem, and I still get no output for any of these genes after running dropEst. Do you have any suggestions for how I might be able to troubleshoot this?

Here's an example read before editing:

RHAJ112762012 16 chr11 32233692 3 6S53M * 0 0 CTTCCTACTCAGGAAGAAACCATGGTGCTCTCTGGGGAAGACAAAAGCAACATCAAGGC EEEAEEAEEEE/EEEE6EEEEEEE<AE//E/EEEE/EEAE/E/EE/EAEAEE/EAAAAA NH:i:2 HI:i:1 AS:i:52 nM:i:0

And after:

RHAJ112762012 16 chr11 32233692 255 6S53M * 0 0 CTTCCTACTCAGGAAGAAACCATGGTGCTCTCTGGGGAAGACAAAAGCAACATCAAGGC EEEAEEAEEEE/EEEE6EEEEEEE<AE//E/EEEE/EEAE/E/EE/EAEAEE/EAAAAA NH:i:1 HI:i:1 AS:i:52 nM:i:0

[potulabe]

As far as I understand, it checks the flag IsPrimaryAlignment, usually secondary alignments are marked with a secondary alignment flag.

[SevenTea7]

Hello, I encountered some problems when installing dropest, and many dependency packages could not be installed. Could you please share this package with me? Thank you very much