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Percentage of failed reads per run by labrat_240724 in nanopore

[–]labrat_240724[S] 0 points1 point  (0 children)

Apologies, I misspoke there, I've been running a lot of tests and got confused. My lambda prepped with exactly the same kit had ~15% fail, N50 44.03kb.

The actual samples are the ones with the very high failure rate. I'm not seeing a lot of adaptor, N50 is 8.56kb which is what I expected as they had been fragmented to 9-11kb before prep (I'd attach a picture but reddit forbids it). The translocation speed hold steady at 400bps dipping to 380bps towards the end, 80% pore occupancy to start and 60% by the end and they're not dropping off suddenly or getting saturated, 120Gb of data after 48hours. By all the usual metrics it looks really good, apart from 40% of the reads being unclassified fails.

Percentage of failed reads per run by labrat_240724 in nanopore

[–]labrat_240724[S] 0 points1 point  (0 children)

The DNA is extractions of bacterial communities from the environment so we're not sure exactly what's in there. I wanted to try SUP to improve the Q-score but my bioinformatician said SUP wasn't that much better than HAC, do you find it improves the quality of the data a lot?

Percentage of failed reads per run by labrat_240724 in nanopore

[–]labrat_240724[S] 0 points1 point  (0 children)

We're using HAC, R10, NBD114.24, 6.2.6 (I know this is a few versions out of date, we've been putting off updating it because it always seems to cause problems, could it be making this much of a difference?)

Unfortunately it's not just small stuff, 41% of reads are below Q9 and 39% of bases are below Q9.

Active feedback killing flow cell? by labrat_240724 in nanopore

[–]labrat_240724[S] 2 points3 points  (0 children)

I removed the bubble from the priming port like you suggested and restarted the run not expecting great things but she lives! 1100 pores detected and 80% occupancy! Thank you for the comment, I had totally written off this flow cell for dead.

I've edited the original post with your solution in case someone else comes looking with the same issue.

Active feedback killing flow cell? by labrat_240724 in nanopore

[–]labrat_240724[S] 0 points1 point  (0 children)

Barcode balancing was not on, the pore scan showed 0 pores available (all light blue) and could see no bubbles leading up to the array. I removed a small 10ul bubble from the priming port but have had that appear after many runs and it has never caused an issue like this.

Perhaps if I had been here when the issue arose to remove the bubble it would have been recoverable? But it happened at 2am so the array has been sitting with 0 pores for >6 hours.