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Old 01-04-2017, 02:26 AM   #1
jordi
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Default Newbie MinION questions

Hi all!
I've some questions about Oxford nanopore Minion technology. I had a deep insight into the community of nanopore but I was unable to find out the answers:
1. What does it mean the "bps" terms of the workflow protocols of Metrichor? Is it just a kind of Internet connection?
2. I've just run the Burn-in protocol. How can I compare the results in order to be sure that the sequencing process ran properly?
3. How should I proceed in order to re-use the flow cell? I mean, the flow cell will have a kind of contamination DNA, won't it?
4. Finally, is there a kind of amplification in the library construction that allows to assembly several reads in order to obtain a larger genomic sequence than those gathered by Minion (I mean larger than 16Kb, for instance).
Sorry if my questions annoyed anyone, but I am still as Illumina-thinker
Thank you for your help in advance.
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Old 01-05-2017, 11:24 AM   #2
Ola
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Hi!
1. This is the sequencing speed. 2D runs at 250 bases/s while 1D now goes through at 450 bps.
2. You would have to post on the forum and ask what others think... It would be great if Metrichor had some kind of minimum specs for yield and quality to compare to.
3. Yes. That is why you got the wash kit. It will not remove everything though.
4. I have no idea what you mean here but there is no amplification in the standard protocol. Of course you can assemble reads in silico afterwards. Canu is probably best for this, with good coverage.
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Old 01-05-2017, 12:16 PM   #3
wdecoster
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About the wash kit, have a look at the following thread on the MinION community forum: https://community.nanoporetech.com/p...oading-with-on
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Old 01-05-2017, 04:11 PM   #4
jordi
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Hi Ola and wdecoster. Thank you very much for your answer, that was definitively helpful.
In the last point I meant the following: if there is no amplification step, how can I assembly a large region? I mean, how a certain region is read several times in order to obtain overlapping reads that allows the assembly? In other words: I got a 1500X coverage of lambda genome in the burn-in protocol. How I got that coverage? I guess I had at least 1500 times the lambda genome, hadn't I?
The following experiment should be a BAC sequencing protocol. Should I amplify the region of interest in order to get a proper number of copies, shouldn't I?
Thank you very much for your help.
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Old 01-06-2017, 06:41 AM   #5
gringer
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You assemble a large region by having multiple DNA copies. Consider a situation where DNA is extracted from a colony of 10,000 bacteria. In an ideal world there would be 10,000 copies of the bacterial DNA floating around in that extract, even without any amplification. Practically, the amount will be considerably less than this, but the recommended protocols use far in excess of this coverage amount.

The standard MinION sample prep requires a reasonable amount of DNA to be present (but not out of the ordinary for high-throughput sequencing): about 200ng for the rapid sequencing kits. This represents an amount on the order of 33k unamplified copies of DNA for human-sized genomes, so there shouldn't be a problem with getting 1000 copies of sequence to cover a particular region, especially if it's bacterial DNA.

Last edited by gringer; 01-06-2017 at 05:50 PM. Reason: updated according to AllSeq's calculations
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Old 01-06-2017, 05:44 PM   #6
AllSeq
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Quote:
Originally Posted by gringer View Post
...about 200ng for the rapid sequencing kits. This represents an amount on the order of 10¹² unamplified copies of DNA for human-sized genomes, ...
Conceptually I agree with your explanation, but surely 200ng doesn't represent 10^12 human genomes. One human genome is ~6 pg. 200ng = 200,000pg, so it's roughly 33k genomes-worth of DNA, right?
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Old 01-06-2017, 05:53 PM   #7
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Sorry, yes, you're right. I had 2pmol in my head as the adjusted quantity for standard 1D ligation prep for 1-8kbp amplicons (it's actually meant to be 0.2pmol), and was confusing that with the 200ng for the transposase kit. I've updated the numbers according to your calculations.
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Old 01-06-2017, 05:56 PM   #8
AllSeq
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Cool. I've been out of the lab for a while and just wanted to make sure I hadn't completely lost it ;-)
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Old 05-30-2017, 03:36 PM   #9
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This counts as a newbie question, I guess, so I won't multiply threads.

1) It states that it is "recommended" to run the phage experiment, and to officially submit the results back to Oxford Nanopore. What are the implications of doing or not doing so? What is that you are forfeiting?

2) If you do choose to sequence the phage, do you have to use the whole flowcell for that?

3) It's also asked that you send the flowcells you have used back. What is the motivation there? Do you absolutely have to do it?

Thank you for all the answers in advance.
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Old 05-30-2017, 03:41 PM   #10
gringer
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Quote:
If you do choose to sequence the phage, do you have to use the whole flowcell for that?
The phage experiment is a short run; you can use the same flow cell for another experiment later on. I'm not sure if the starter pack includes a wash kit, but you can order one from ONT (currently $189 for 12 washes):

https://store.nanoporetech.com/catal...9/category/28/

The other questions are best directed at ONT. I suggest you email them via <[email protected]>.
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Old 06-01-2017, 07:42 AM   #11
lorendarith
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Quote:
Originally Posted by gringer View Post
I'm not sure if the starter pack includes a wash kit, but you can order one from ONT (currently $189 for 12 washes):
The starter pack contains a wash kit.
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Old 06-01-2017, 07:46 AM   #12
gringer
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Thanks. I'll try to remember to check the website before making statements like that again.

https://store.nanoporetech.com/minion/sets
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