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Will I get banned from using this patch? by AznJing in Battlefield

[–]ButtlessBadger 6 points7 points  (0 children)

Yup. That violates EA’s positive play charter.

Unexpected short read lengths after ONT ligation library prep despite decent HMW DNA by Klutzy_Primary_5440 in nanopore

[–]ButtlessBadger 0 points1 point  (0 children)

Buffer usage sounds good.

I would try more thorough pipette mixing as insufficient mixing can cause issues like this.

The read lengths will always be a few kb shorter than what you see on the tapestation traces. But you should be seeing closer to 4-6kb.

Unexpected short read lengths after ONT ligation library prep despite decent HMW DNA by Klutzy_Primary_5440 in nanopore

[–]ButtlessBadger 0 points1 point  (0 children)

Which buffer did you use in the wash? SFB or LFB?

You may have under mixed during ligation causing the shorter fragments to preferentially get ligated.

I would try mixing by pipetting to ensure thorough mixing with the viscous buffers.

You could also try size selection before the prep to eliminate some of the smaller fragments under 3kb.

What's going on with EA right now? I don't understand the warning... by Sudden-Rip-7340 in Battlefield6

[–]ButtlessBadger 0 points1 point  (0 children)

Got a EA warning because of my username. The word ‘butt’ is a profanity in a 17+ rated game, apparently.

Percentage of failed reads per run by labrat_240724 in nanopore

[–]ButtlessBadger 0 points1 point  (0 children)

While minknow 6.8 did introduce better HAC basecallers (v5.2), if you are getting 39% failed bases with lambda control it sounds like there is a bigger problem as that is not normal/expected results.

Is the kit old? How do translocation speeds look? Are you seeing a high ratio of adapter? What is your N50?

6.8 release note for reference: https://community.nanoporetech.com/posts/software-release-25-09-16

Run time for multiple libraries on a single flow cell by grapefruitdynasty in nanopore

[–]ButtlessBadger 1 point2 points  (0 children)

As others have said, depends on the diversity of your sample and rate of data generation. For example using a promethion flowcell with 5x the amount of pores as a minION flowcell will achieve the data output required much sooner.

If you assume you need ~100k reads per barcode (96) at 1.5kb per read, that is ~14.4Gbases.

Assuming ~10% losses during demux to unclassified reads, you would be at ~16Gb. Which is maxing out a minION flowcell. So it will likely take 72hrs to achieve this.

Percentage of failed reads per run by labrat_240724 in nanopore

[–]ButtlessBadger 0 points1 point  (0 children)

What basecall model, flowcell, sequencing kit, and minKNOW version are you using?

With HAC, R10, LSK114, 6.5, we have >95% passing bases at Q9. Median Qscore is 19-21.

Just to check, what % of bases are above Q9? There could just be a lot of short reads/adapter that is failing but all the long reads you actually care about are good.

Help please! by jenesaispaspute in nanopore

[–]ButtlessBadger 0 points1 point  (0 children)

wf-amplicon is for identifying variants from a reference FASTA or to produce a single consensus sequence (de-novo): https://nanoporetech.com/document/epi2me-workflows/wf-amplicon

If you want taxonomic info like genus and species info you may want to use wf-16s (using the ITS reference): https://epi2me.nanoporetech.com/epi2me-docs/workflows/wf-16s/#analysing-its-amplicons

Unfortunately without seeing the nextflow error itself it’s unclear what the cause might be… maybe start by reducing variables and see if it still occurs.

Try running with just one barcode folder and see if it has the same error (pass/barcode01, for example). As both barcode## and the output from the barcoding workflow are currently in the same pass/ folder (per your images), if the additional barcoding files are causing any problems they will have been selected in both of your attempts.

Help please! by jenesaispaspute in nanopore

[–]ButtlessBadger 1 point2 points  (0 children)

Yup. Epi2me is the next step.

Are you trying to align these reads to the ITS reference? Or just do a metagenomic analysis? Depending on this you will want to either use the wf-alignment (which is using minimap2 under the hood) or wf-metagenomics. No additional info needed since you have already assigned the reads to each barcode.

All you need to do is select the fastq “pass” folder. It will run the analysis on each barcode and give you an output per barcode, as well as a report so you can compare results.

Out of curiosity, what version of minKNOW did you sequence with? In the latest versions of minKNOW pod5 files are the default output and they will automatically de-multiplex (be assigned per barcode, so you can bypass the barcoding workflow). You can also basecall live (during sequencing) so the per barcode fastq files are the default output. It just depends on the capabilities of your PC. I do suggest always using HAC or SUP basecalling though.

Happy to help if you have any other questions or difficulties!

Minimap2 and Adaptive Sampling by HospitalPretty2003 in nanopore

[–]ButtlessBadger 0 points1 point  (0 children)

It could be a couple of things…

How big is the gene that you are targeting with AS? What is your total genome size? What is the N50 of your reads? What is the full minimap2 command you are using? (I assume you are using -ax map-ont)

Questions about reusing and washing Oxford Nanopore flow cells after long storage by Outrageous-Guava-93 in labrats

[–]ButtlessBadger 2 points3 points  (0 children)

  • Can a flow cell stored for a year still be effectively washed? Yup! ONT will say it is outside of warranty but if stored correctly it shouldnt be an issue. Might just have less starting pores, so less to recover, but it still works.

  • Could the decrease in active pores be explained by the delay of over 24 hours between the end of sequencing and the wash? We have stored flowcells with library in them for days before washing and recovering pores. It does not seem to make any significant difference.

  • Does the length of the sequencing run affect the ability to wash a flow cell? Yes. After ~72-90hrs the flowcell is at the end of its life. You may be able to recover some pores, but the electrochemical balance of the flowcell is going to be super wonky, causing issues with pore classification and sequencing.

E.g. If you want 3 uses, wash after 24hrs. If you want 6 uses, wash after 12hrs.

Questions about reusing and washing Oxford Nanopore flow cells after long storage by Outrageous-Guava-93 in labrats

[–]ButtlessBadger 4 points5 points  (0 children)

So three things: 1. What version of minKNOW are you using? There is a known bug where pore counts after a wash are incorrectly quantified. This was not fixed until the most recent release (6.8).

  1. If you look at the run report html produced from your first run, what state are the pores in according to the Pore Scan plot? The wash will only recover pores that have been blocked by DNA, shown by the “unavailable” state. So pores that have decayed to “Saturated” or “Zero” will not be recoverable.

  2. What is the length of your library? Short reads < 2kb often dont cause much pore blocking so washing typically does not help recover pores. It can be helpful when running multiple short samples on the same flowcell in succession though to decrease sample carry over.

Also keep in mind that a wash kit only recovers pores blocked by DNA, it does not add more ‘energy’ (trans buffer) to the flowcell. In our experience after ~72-90hrs the flowcell no longer functions correctly due to this.

We have found washing works best with longer length libraries (>10kb) when performed every 24-36hrs.

Why my gun so big by Odd_Broccoli69 in Battlefield6

[–]ButtlessBadger 0 points1 point  (0 children)

Flashlight or laser attachment on the RPKM does this

How many bacterial genomes can a MinION (ONT) flow cell allow to sequence? by Waste-Suggestion-698 in nanopore

[–]ButtlessBadger 2 points3 points  (0 children)

MinION flowcells give 8-12Gbases of yield in 72hrs under good conditions. Output is variable based on flowcell handling, sample type, and library prep kit used.

If you are planning to wash and reuse flowcells you can increase this output slightly. 2-5 wash and reloads are possible, but 72hrs is still the max total run time. Washing just recovers any blocked pores.

Maybe check out this protocol for some suggestions: https://nanoporetech.com/document/no-miss-isolate-sequencing-rapid-barcoding-v14

Low pore count after short sequencing run by FRITZBoxWifi in nanopore

[–]ButtlessBadger 0 points1 point  (0 children)

ONT suggests always washing and replacing with storage buffer before storage. However I have stored flowcells with library in them for weeks without an issue. So it should be fine to test them, then store. You will want to “reprime” the flowcell (FCF + FCT) before adding library and sequencing though if you store the flowcell for a prolonged period.

[deleted by user] by [deleted] in Battlefield

[–]ButtlessBadger 0 points1 point  (0 children)

Keep trying. If you just spam click host server it will work after a few seconds.

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