We've just started using the Illumina Next-Seq platform and haven't been getting good results sequencing our CEL-Seq2 library (75cycle, R1=15bp, R2=77bp), as opposed to what we've been getting previously using Hi-Seq.
QC apparently "looks fine". But I'm not convinced given that the deviation bars in FastQC are huge.
%Q30 is ~78%.
The problem is that %mapping to the reference genome is only ~20%, even after discarding sequences with ave Qscore<30.
Tech advised that we've probably overloaded the DNA.
We paid for another run of the same library at 30% less loading (~0.9pM) and 20% phiX-spike in.
The same problem persists. %Q30 is better at 88%, but again, the sequences are full of N's (20% of R1 sequences are all Ns) and map at 20%.
We've been told it could be the library. But there are definitely enough DNA at ~200-400bp size.
I'd like to get an opinion on whether this is likely a library-prep problem (old reagents?) or sequencing-problem (settings?)...? What could be the triggering issue?