Hi All 

I have some samples with low mapping in Salmon (40% and less) that have higher alignments in Tophat, and trying to troubleshoot. 

I picked some of the unmapped reads (from writeunmapped salmon parameter) and Blat them to human. 

Some have 2 or more matches with identity 99% to 100% 
And some have many many matches, I need to scroll the page down too much. Many of these matches are 100% and some range between 85% to 100% identity. 


I looked also into the “ambig_info.tsv’ , found some records with 0 unique mapping and more than 100 ambiguous mapping, but couldn’t relate to those unmapped. 

This is how one match of one of them look in one mate and the other: 

     
    ACTIONS      QUERY           SCORE START  END QSIZE IDENTITY CHRO STRAND  START    END      SPAN
    
    browser details YourSeq           22   100   122   151 100.0%    10   +   37378275  37378303     29
    browser details YourSeq           22    52    74   151 100.0%    10   -   37378275  37378303     29

So why this is not counted as mapped for example?  Any hint, clue?  

Thanks
Salmon maps reads to the transcriptome while TopHat aligns to the genome (assuming that you are using Tophat in the "normal" way). I suspect that if you examine the reads that map with TopHat but not with Salmon, some (perhaps large) proportion of them is mapping to intergenic and/or intronic regions, so those reads will not be mapped by Salmon. Taking a look in a browser at the BAM file from TopHat or using a tool like RNASeqQC can help quantify the intergenic/intronic reads. 