Entering edit mode
10.6 years ago
mazzottidr
▴
30
Dear all, I've just got some Illumina exome data from MiSeq (Nextera capture) and got the following fastqc results. Would someone give feedback on what might be wrong (if so)? Would trimming help in this case? Are these indicative of barcodes or adaptors?
Thank you all in advance!
What do you see in the "Overrepresented Sequences" section of your FASTQC report? Also, those images are too small (for me, at least) to interpret.
Thank you for the reply. No flag was found on Overrepresented sequences... Here is the link for full size images https://github.com/mazzottidr/questions/tree/master/M132N_S1_L001_R1_001a_fastqc/Images Are those graphs indicating some other problems with my files?
You can have a look at http://pathogenomics.bham.ac.uk/blog/2013/04/adaptor-trim-or-die-experiences-with-nextera-libraries/ The Blog post describes what it looks like if you have adapters in your reads. from my expirence if the reads went through the adapter, you will have long streaks of As in your data after the adapters. This is not the case in yor data, but your kmers look definetly wired.
Thanks... I am gonna check it... I've noticed lots of As and Ts... In any case, I've aligned those reads and despite the low coverage, >95% of reads mapped... Since the read quality looks ok, I guess I can go on... Any more thougths:
Another question: you said you ran these on MiSeq: usually trimming is done automatically on the MiSeq workflows. What kind of run is specified on your sample sheet?
The sequencing center just told me that the barcodes and indexes were removed already, but do you believe those flags on fastqc qould compromise frther analysis?
I don't want to post this as an answer because I'm not totally confident, but that initial set of spikes in your kmer profile screams "adapter contamination." The problem is that I can't reconstruct a sequence from those first four spikes.