When trying different values for the mate-inner-dist parameter in TopHat v1.3.3 to align a paired end (2 x 76 bp) sequencing run from Illumina, I confusingly notice that this parameter has no effect on the alignments.
I first tried to derive the median insert size directly from sequencing data using the approach described here: http://seqanswers.com/forums/showpos...62&postcount=3
and ran TopHat with the following parameters:
Yet, when performing the analysis again with different values of the mate-inner-dist parameter (150, 50), then comparing the accepted_hits.bam results file using samtools flagstat command, I get exactly the same regardless of this parameter. In particular, properly paired reads remain around 65%.
From what I can read in another post (http://seqanswers.com/forums/showthr...roperly+paired), and based on a my own digging into TopHat source code, I would expect this parameter to affect the number of reads flagged as properly paired.
Is there any explanation? Do you have similar observations?
I first tried to derive the median insert size directly from sequencing data using the approach described here: http://seqanswers.com/forums/showpos...62&postcount=3
and ran TopHat with the following parameters:
Code:
--mate-inner-dist 136 --mate-std-dev 40
From what I can read in another post (http://seqanswers.com/forums/showthr...roperly+paired), and based on a my own digging into TopHat source code, I would expect this parameter to affect the number of reads flagged as properly paired.
Is there any explanation? Do you have similar observations?
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