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Old 10-06-2015, 07:39 PM   #21
Brian Bushnell
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Isn't that just a chemistry thing though? PacBio is a sequencing-by-synthesis method after all, and I don't see any reason why Circular Consensus Synthesis couldn't be done on nanopore as well by generating the sequence prior to putting it through the sequencer.
No... nanopore sucks the reads through like spaghetti. They are read once. 2D reads are achieved by reading a sequence and its reverse-complement. There is no provision for reading any sequence more than twice, because they are not circular.

If there was some way to make replicates of a read linked together in a giant molecule, then yes, Nanopore would be able to make CCS reads. But such a thing is not possible today, or in any claimed future developments.
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Old 10-06-2015, 07:57 PM   #22
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If there was some way to make replicates of a read linked together in a giant molecule, then yes, Nanopore would be able to make CCS reads. But such a thing is not possible today, or in any claimed future developments.
How about attaching hairpins to each end of a double-stranded sequence, then using a strand-displacement polymerase to bind to a primer sequence and run around in a loop, generating a single long sequence that repeats the initial template multiple times:

https://flxlexblog.wordpress.com/201...us-sequencing/
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Old 10-07-2015, 05:20 AM   #23
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According to GenomeWeb, Sequel has a list price of $350,000, half that of a RSII (and a physical footprint a third the size of a RSII).
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Old 10-07-2015, 09:18 AM   #24
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If there was some way to make replicates of a read linked together in a giant molecule, then yes, Nanopore would be able to make CCS reads. But such a thing is not possible today, or in any claimed future developments.
RCA and LAMP is certainly possible today. Maybe not practical, or needed if accuracy goes up.
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Old 10-09-2015, 01:19 AM   #25
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This is very close to the current cost of MinION sequencing. It wouldn't surprise me if that weren't a coincidence.

A great MinION run will currently put out about 1Gbp of sequence, but that will change substantially after fast mode kicks in (about 20x sequencing speed). I wonder how flexible PacBio are with their pricing for the chip and reagents.
But for de novo assembly, only ONT 2D reads can be used. That means usable throughput for de novo assembly is only about 20%. Therefore, even if it gets the 20GB upgrade, the usable throughput is only 4GB. If the pricing remains $1000 per box, it is still not competitive not to mention the lower accuracy.

Of course, ONT is the only game in town for the portable market. I think that will be where its niche is until it can improve accuracy significantly.
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Old 10-09-2015, 05:32 AM   #26
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But for de novo assembly, only ONT 2D reads can be used. That means usable throughput for de novo assembly is only about 20%.
Your information is a little old. The new reagent kits get about 80% 2D reads by using a sample preparation process that binds the hairpin to streptavidin beads.

While 1D reads are not particularly useful now, I have hope that a bright mathematician or computer scientist from outside ONT will substantially improve the base calling model. That will allow all those "useless" 1D reads to be retroactively recalled with much improved accuracy in the future.
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Old 10-09-2015, 05:38 AM   #27
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If the pricing remains $1000 per box, it is still not competitive
It's currently $500 USD per flow cell if ordering in bulk, with reagent cost ~$200 per run, so basically the same marginal cost as the Sequel (ignoring initial equipment cost).

There's a new flow cell design coming out in the next 6-12 months with six times the number of pores and a substantially lower marginal cost, currently projected to be $20 USD for a short 3-hour run producing 2Gbp. I expect that would mean about $100 to run the flow cell for as long as it can go.

Last edited by gringer; 10-09-2015 at 05:41 AM.
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Old 10-09-2015, 06:10 PM   #28
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It's currently $500 USD per flow cell if ordering in bulk, with reagent cost ~$200 per run, so basically the same marginal cost as the Sequel (ignoring initial equipment cost).

There's a new flow cell design coming out in the next 6-12 months with six times the number of pores and a substantially lower marginal cost, currently projected to be $20 USD for a short 3-hour run producing 2Gbp. I expect that would mean about $100 to run the flow cell for as long as it can go.
In most places to get funding for a project one must estimate costs based on available technologies with predictable outputs. Cost estimation based on promised outcomes from technology providers with unknown real costs would result in rejection by fund providers. ONT currently has no definitive output and quality specs making it interesting to some bioinformaticians who see a fresh ground for software developments. It is also notable that non-optical based sequencing technologies has been less successful so far.
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Old 10-10-2015, 07:04 AM   #29
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I was at ASHG on Wednesday and Thursday and the PacBio booth sure was VERY busy.
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Old 01-28-2016, 12:26 AM   #30
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If there was some way to make replicates of a read linked together in a giant molecule, then yes, Nanopore would be able to make CCS reads. But such a thing is not possible today, or in any claimed future developments.
Following on from this, there has been a recent preprint demonstrating a very similar approach for ONT sequencing:

http://biorxiv.org/content/early/2016/01/27/038042

[I hope the authors don't mind sharing links to this]
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Old 06-16-2016, 11:55 PM   #31
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I wonder if anyone have update on Sequel performance and how it compares to RSII.
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Old 06-17-2016, 03:10 AM   #32
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There was a PacBio user group meeting in Baltimore (in last two weeks) where an update was given about Sequel performance (based on a note I saw from a FAS). Apparently that information has not been published but if someone on SeqAnswers was in attendance then they may be able to comment.
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Old 06-17-2016, 04:24 PM   #33
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Thanks GenoMax. The system release was announced almost 8 months ago and I thought there might be some installed machines in production or in validation phase where some operators might have been trained and seen some real data to compare with Sequel announced specs or RSII.
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Old 06-17-2016, 07:44 PM   #34
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Thanks GenoMax. The system release was announced almost 8 months ago and I thought there might be some installed machines in production or in validation phase where some operators might have been trained and seen some real data to compare with Sequel announced specs or RSII.
We don't have any data, but we have a (growing) list of service providers who have installed the Sequel platform: http://ngs.is/PBSequel
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Old 06-18-2016, 05:03 AM   #35
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They do not seem to offer any service on the Sequel.
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Old 06-18-2016, 05:27 AM   #36
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Thanks GenoMax. The system release was announced almost 8 months ago and I thought there might be some installed machines in production or in validation phase where some operators might have been trained and seen some real data to compare with Sequel announced specs or RSII.
This is a big mystery. There are reports of machines in the wild (at least with close collaborators of PacBio) but no one seems to have released real data as yet. There is nothing EBI-ENA at this time either.
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Old 06-18-2016, 09:13 AM   #37
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We have two of them, but they are still being qualified.
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Old 06-18-2016, 10:46 AM   #38
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A few service providers had told me they expect to start providing Sequel services in June. I expect we should see some data soon?

At the UGM pacbio did mention they expect to start shipping their new chip at the end of the month along with a new loading protocol. So maybe service providers are waiting for that? I'm sure Sequel owners here would have a better idea of whats going on though.
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Old 06-20-2016, 09:33 AM   #39
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Hi Brian,
is this with your previous or the new employer?
Thanks!

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We have two of them, but they are still being qualified.
A Sequel talk from April: https://www.youtube.com/watch?v=hmx3HGs9ims

Last edited by luc; 06-20-2016 at 09:46 AM.
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Old 06-20-2016, 09:45 AM   #40
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Hi Brian,
is this with your previous or the new employer?
Thanks!


Both! I left Google and came back to JGI. So, it's at JGI.
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