Yet again, I’m faced with another person in our building coming with to me with those god damn reads using 27F 519R primers with the v3 (2x 300bp chemistry). Yes, I’ve now passed on messages to our sequencing centre and yes to the school as well about not going down this road. ANYWAY, is there a possible solution to tweak make.contigs to better handle this situation. The guys in the sequencing centre said:
"There are some reagent performance issues with the v3 600-cycle kits from Illumina which manifest as poor quality towards the end of each read of a long read run (2x300bp), but particularly affect read 2. The last update I had from them indicated they may have found a cause, but are still investigating. They have not put any usage or purchase holds on these reagents and have not issued any technical bulletins or warning about them either. One reason for this is that there is variability in the performance of the reagents even from the same lot, and runs can still pass their performance parameters, although some more stringent mitigations are now necessary whereas previously these were not much of a concern. These include lowering the cluster density and increasing the PhiX spike in.
and passed on some info that another group was doing;
“The quality of all illumiuna R1 and R2 reads was assessed visually using fastqc . Generally we observed a significant drop in read quality in the last 50-100bp of R2 and the last 10bp of R1. We trim the 5’ end of R1 by 10bp and the 5’ end of R2 by 70bp (we chose to trim as many bp as possible while
still leaving an overlap that allowed reliable merging of R1 and R2 reads. Reads were then merged using FLASH . After merging several hundred sequence were merged manually and the results compared to the FLASH merges to ensure efficacy of FLASH.”
I’m naturally curious and will have a look at this approach. I’m most interested in whether trimming the sequence before attempting to make contigs will aid in not losing ~ 60 % of your sequences after screening contigs!!! :shock: (generally due to at least 1 or more NBases being present) and also keeping in mind that those 40 % that pass, 30 % of them (30 % of the 40 %) are unique :lol: Garbage in garbage out…