Dear all,
Currently, I am processing a NGS data using cutadapt to remove 3p and 5p adapter sequences from reads. Normally, as I notice, the percentage of trimmed reads (reads with removed adapters) could reach 95 - 99 %. But for this data, some of my sample only contain 20 - 25 % reads with adapters.
What could be the reasons for this phenomenon? I have carefully check the cutadapt command and everything is fine. Thanks!
You can double check whether 'cutadapt' removes adapters sequence only or otherwise it implies some other quality filters such as average Phred quality score along sliding window of 4, 5, or 6 bases, and minimum length of reads after adapters clipping etc. This depends on default parameters.
Second, you can check what was the average size of your DNA library, and what is the length of your sequenced reads. If you have got higher read length (such as 150x2 or even higher) for a shorter sized library, you will get a lot of adapter sequences in the final data which will ultimately be removed in adapter trimming step, leaving behind less percentage of adapter-free filtered reads.
maybe the truth is somewhere else....
it sounds like ligation during library preparation has not been properly done.
fred
You can try NGS-QC toolkit for quality filtering and adapter trimming. I am attaching the manual and link to download below. Hope it may help you.
http://www.nipgr.ac.in/ngsqctoolkit.html
- Badges
- Science method