Entering edit mode
6.5 years ago
Zac Shi
•
0
I got a strange distribution of insert size as analyzed by Qualimap on the BAM file. Not totally aware of the library preparation procedure yet, but this is a PE-sequencing on NextSeq500, libraries were made with NEBNext Ultra II kit and size selection beads. The median insert size was expected to be around 300bp. The problem is that we got not only a much smaller median insert size (190bp), but also a non-symmetrical distribution. What could be the reason? Is this a concern? Thanks for any help. 
I suspect your read-length is ~135bp? The insert size is usually the outward distance so the lower bound is the read-length. Alfred (disclaimer: my own tool) splits the insert size by all possible 4 types of read pair orientations so this will at least tell you if you have the standard illumina PE layout. In general, shorter fragments cluster better so it is not unusual that the insert size shifts to smaller values in-silico compared to what you see on the Bioanalyzer (and the Bioanalyzer plot is with adapters).
no offense but this question is more a wet lab question than a bioinformatics one.
Thanks for the reminder. It does sound like that. So I just put it on the other forum too, http://seqanswers.com/forums/showthread.php?p=217166#post217166 . Hopefully someone here or there could help.