Entering edit mode
10.2 years ago
DoubleDecker
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180
Is there any tool that can help with ambiguous (multimapping) reads in BWA - assign them to more probable transcript based on such things e.g. as mapping depth. We have RSEM, express but they all for some reason seem to be geared towards Bowtie... so is there any other solution?
I tried eXpress but it does not seem to count the reads correctly, summed counts total is significantly lower than what I get when I look at output from samtools flagstat ran on the same reads but mapped without using the -a option in bwa-mem.
Did you try using the same bwa-mem options to compare both methods?
Well, yes, I ran samtools and express on the same input file. In fact, I had a look at express mailing list where they admit that express is not compatible with BWA yet. You can find a script there which should be able to convert bwa-aln output to bowtie output and thus work with both RSEM and express but again, it does not work with bwa-mem.
Hummm... that's surprising. They should state it on their website/documentation if they are aware of this incompatibility. All they say now about the output is "eXpress also requires a file, multiple files, or a piped stream of SAM or binary SAM (BAM) alignments as input. The SAM alignments should be generated by mapping your sequencing reads to the target sequences specified in the multi-FASTA input file described above. For more details on the SAM format, see the specification. Many short read mappers including Bowtie, Bowtie2, BWA, and MAQ can produce output in this format."
I also compared HTSeq-count and eXpress and the results (read counts) are very different. Not sure which I should trust... I get in total 35% less reported reads with htseq but some of them are higher, some are lower. This is very strange taking into account that I'm using the same SAM files.
I asked the question on another forum since nobody here seems to have an answer... (sorry about that)