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Oxford Nanopore MinION – USB stick-sized DNA sequencer

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Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#21

Post by mbreese »

njbooher wrote: post #20
mbreese wrote: post #17

Earlier quoted context omitted.

Actually PacBio does just that to get better a better error rate. They basically have X amount of sequencing that can be done. You can spend that X however you'd like. If you want a sequence that is X long, you'll have a higher error rate. If you want a chunk that is X/10 long, you can circularize it, and thus sequence it ten times. This gets you better accuracy with the redundancy. DNA is pretty robust. In practice…

I don't think they push CCS much anymore. The focus seems to be on generating long reads and then using Quiver to call the consensus ( https://github.com/PacificBiosciences/GenomicConsensus/blob/... ).

That's probably wise of them. The last time I looked at data from them, the CCS reads were still very noisy. It's better to rely on your strengths. In their case, it's long reads.

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#22

Post by dkural »

ihodes wrote: post #16

The error rate of 15% (I actually believe it's more like 30% in practice, which is 2x PacBio's) is actually excellent and comparable to PacBio's in that the error is primarily randomly distributed, and not systematic like current Illumina or IonTorrent systems (which have admittedly lower error rates, at 1-3%). The short of it is, absolute error rate is not the whole story. When looking at what ends up being just a m…

I entirely disagree. Illumina actual error rates (as opposed to base quality) is less systematic than current Oxford Nanopore reads, which there are admittedly very few in public.

Pac Bio SMRT kits are super cheap and highly multiplexed (ie produce a lot more data) in comparison to Oxford Nanopore. The error rates are lower, average read lengths higher.

I don't see any large market so far for ONP. Of course with better data and lower error rates things might change..

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#23

Post by njbooher »

mbreese wrote: post #21
njbooher wrote: post #20

Earlier quoted context omitted.

I don't think they push CCS much anymore. The focus seems to be on generating long reads and then using Quiver to call the consensus ( https://github.com/PacificBiosciences/GenomicConsensus/blob/... ).

That's probably wise of them. The last time I looked at data from them, the CCS reads were still very noisy. It's better to rely on your strengths. In their case, it's long reads.

Yup. Just keeps getting better (https://pbs.twimg.com/media/BpUs_MAIYAEHb2o.png:large).

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#24

Post by dekhn »

dnautics wrote: post #10
dnautics wrote: post #8

The error rate in the "good part" is about 15%. That is quite frankly very very very poor. This might be a good alternative to pacbio, (unless the other technologies can increase their read lengths). Typically (I chatted with my contacts at the Venter Institute)... best results are gotten by making long reads using pacbio or (maybe minion, they are looking into that currently) and using that to generate the scaffold…

More information: For personal (as in human genome) sequencing, I'm not entirely sure how useful this is... We already have a scaffold for the human genome (that would be Mr. Venter himself) and MAYBE you could get some haploid information out of it, but you'd worry that the base pair you care about for any given individual is in one of the wrong stretches. For organism sequencing, I think this could be even worse th…

Is there an accuracy problem in PacBio's published de-novo assembly human genome: http://blog.pacificbiosciences.com/2014/02/data-release-54x-...

it was assembled de-novo (IE, without HuRef), and the accuracy stats aren't horrible.

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#25

Post by owlmonkey »

dekhn wrote: post #24
dnautics wrote: post #10

Earlier quoted context omitted.

More information: For personal (as in human genome) sequencing, I'm not entirely sure how useful this is... We already have a scaffold for the human genome (that would be Mr. Venter himself) and MAYBE you could get some haploid information out of it, but you'd worry that the base pair you care about for any given individual is in one of the wrong stretches. For organism sequencing, I think this could be even worse th…

Is there an accuracy problem in PacBio's published de-novo assembly human genome: http://blog.pacificbiosciences.com/2014/02/data-release-54x-... it was assembled de-novo (IE, without HuRef), and the accuracy stats aren't horrible.

PacBio is ultimately more accurate than Illumina given consensus. Illumina has some systemic bias so the ultimate accuracy tops out around Q40-Q50 (same errors just repeat themselves) and PacBio will get you closer to Q60-Q70 because the random errors resolve out. So it's the most accurate platform actually. This singuler MinIon read however was barely mappable to the reference, so it looks pretty poor quality. But without more reads we don't quite yet know what the systemic error profile will be though for it. That's why they're adding more pore types, they say, to get multiple systemic error profiles to try to cancel some out.

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#26

Post by owlmonkey »

mbreese wrote: post #17
petercooper wrote: post #13

Earlier quoted context omitted.

Naive idiot alert.. could you process the "same" DNA numerous times then take the mode in each case? Or are the errors essentially ones that would be repeated each time?

Actually PacBio does just that to get better a better error rate. They basically have X amount of sequencing that can be done. You can spend that X however you'd like. If you want a sequence that is X long, you'll have a higher error rate. If you want a chunk that is X/10 long, you can circularize it, and thus sequence it ten times. This gets you better accuracy with the redundancy. DNA is pretty robust. In practice…

One doesn't need CCS anymore, though it is still an option for shorter insert lengths. The errors are more random than other platforms, so they resolve easily with consensus. You just need 20x coverage or more, depending upon the application. With enough coverage you generally get better accuracy than any other platform because it's the least biased technology.

CCS still works really well if you want incredibly high accuracy. See this paper and figure 3 for Q90 quality reads: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811116/

But most people don't need that for their applications. So just regular consensus using PacBio data alone is sufficient for excellent (Q60 or better) consensus accuracy: https://github.com/PacificBiosciences/GenomicConsensus/blob/...

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#27

Post by owlmonkey »

pcrh wrote: post #18
ihodes wrote: post #16

The error rate of 15% (I actually believe it's more like 30% in practice, which is 2x PacBio's) is actually excellent and comparable to PacBio's in that the error is primarily randomly distributed, and not systematic like current Illumina or IonTorrent systems (which have admittedly lower error rates, at 1-3%). The short of it is, absolute error rate is not the whole story. When looking at what ends up being just a m…

As it stands, it looks good enough for identification purposes (for example of a pathogen), but nowhere near good enough for genetic studies where single base pair variations are the most sought-after aspect. For human genetic studies, I guess it also depends on how readily multiple reads can be obtained, and whether the errors are randomly distributed.

Totally agree. It is at best a semi-portable sensor (with wet prep) not a sequencer. Cost would be another big obstacle for using MinIon on human data. At about 100 megabases and $1000 per cell that's three orders of magnitude more expensive per base than a HiSeq X. (edit: spelling)

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#28

Post by owlmonkey »

dnautics wrote: post #10
dnautics wrote: post #8

The error rate in the "good part" is about 15%. That is quite frankly very very very poor. This might be a good alternative to pacbio, (unless the other technologies can increase their read lengths). Typically (I chatted with my contacts at the Venter Institute)... best results are gotten by making long reads using pacbio or (maybe minion, they are looking into that currently) and using that to generate the scaffold…

More information: For personal (as in human genome) sequencing, I'm not entirely sure how useful this is... We already have a scaffold for the human genome (that would be Mr. Venter himself) and MAYBE you could get some haploid information out of it, but you'd worry that the base pair you care about for any given individual is in one of the wrong stretches. For organism sequencing, I think this could be even worse th…

Ya the MinIon is more of a DNA sensor than a sequencer in its current incarnation, but awesome for portability. Super expensive per base though. If they can't improve the cost significantly along with the accuracy it will never be a competitive sequencer.

You're wrong about the PacBio though, the raw reads are random and cancel out easily with consensus. PacBio is the least biased and therefore the most consensus accurate next-gen sequencer available. See my other comments for some citations.

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#29

Post by owlmonkey »

rflrob wrote: post #3
facepalm wrote: post #2

What does it mean? I don't suppose I can put a drop of blood onto that sequencer and receive a sequence?

In theory you could, at least according to the hype. You would probably be better off taking a drop of blood, mixing with various reagents to lyse the cells and lightly fragment the DNA, but that's the dream. Of course, thanks to the accelerated Moore's law that applies to sequencing over the last couple decades [1], it'll probably be the case that the computer power will be more expensive than the actual sequencing.…

Totally agree with you on the computation costs.

In some slides in February ONT hinted at why they had to go to a full wet sample prep and it related to input DNA amounts I believe. Their new sample prep they said allowed them to reduce their input DNA amounts to similar amounts as competitor sequencers, but without that new prep it took 10,000x more DNA to load. I guess raw DNA just doesn't load into the nanopores very easily, so it took that much higher concentration. Two years ago they claimed you could load raw blood into a chip after a 5 minute prep, but I'm guessing they will back away from that claim now since this wet chemistry is needed to get reasonable loading.

This to me implies that all nanopore technology under development will encounter similar problems. They really need biological or magnetic bead processes to "load" DNA through the pores in one form or another - or else need prohibitively high DNA concentrations and amounts - so that will mean wet chemistry.

Re: Oxford Nanopore MinION – USB stick-sized DNA sequencer

#30

Post by tkinom »

kanzure wrote: post #19
tkinom wrote: post #6

Very nice! I worked on the firmware for the "Applied Biosystems ABI Prism 310 DNA Sequencer". I remembered it cost >$60K each when it was released ~1996, 1997 time frame. It is good to see DNA sequencer tech progress faster than Semiconductor.

> I worked on the firmware for the "Applied Biosystems ABI Prism 310 DNA Sequencer". Do you have copies of that firmware or know where I could pick it up? I'd like to poke around, try some reverse engineering, etc.

Sorry, it was not open source. BTW, it is just standard embedded firmware that control 3 axis motors + pump, CCD camera, etc. Highly couple to that particular instrument' HW. I am not sure what you can learn from it even if it was open source.
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