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Old 08-11-2019, 01:01 AM   #16
gringer
David Eccles (gringer)
 
Location: Wellington, New Zealand

Join Date: May 2011
Posts: 836
Default Hobby Money

Quote:
Originally Posted by Haiqu View Post
In any case, I can't see any valid reason to be paying return shipping to the UK if the device is merely going to be disposed of.
Customers don't pay return shipping to the UK. That cost is met by ONT, even for flow cells that are being returned from New Zealand. I suppose if you want to be cynical, you could say the return shipping cost is included in the price of the flow cells.

Quote:
I can't see a hobbyist needing to handle the volumes for 12-sample multiplexing, and the learning curve for barcoding would complicate an already complex procedure.
You can either multiplex and reduce the per-sample cost, or not multiplex and get a higher yield per sample. Flongle flow cells ($100 USD each, for about 1/10 of the yield) make that decision a bit more palatable. There's essentially no learning curve for rapid barcoding on top of standard rapid sample prep. Here's the normal sample prep:
  1. Add 2.5μl fragmentation mix to 7.5μl sample in a sample tube
  2. Heat to 30C for 1 min, then 80C for 1 min
  3. Add 1μl adapter mix, Wait 5 mins
  4. Mix with sequencing buffer and load onto flow cell

Here's the barcoded sample prep:
  1. Add 2.5μl barcoded fragmentation mix to 7.5μl of each sample in a sample tube
  2. Heat to 30C for 1 min, then 80C for 1 min
  3. Mix all the prepared samples together
  4. Put 10μl into a new tube
  5. Add 1μl adapter mix, Wait 5 mins
  6. Mix with sequencing buffer and load onto flow cell

Sure, it's two more steps, but it doesn't involve any more learning.

Quote:
A more likely scenario is use and rewash of the flow cell, where maybe 4 or 5 samples can be read at best.
Yes, that can be done as well. ONT provide a wash kit for $190 that works for 12 rewashes, but people have discovered (with ONT's help) that a nuclease flush is actually more effective (carryover with a nuclease flush is something like 0.01% vs 1-5% with the wash kit).

It works. Depending on how much yield you want, a flow cell can be washed and re-used for 8 runs, probably more.

However, the flow cells contain ions that pass from one side of the membrane to the other, and these deplete over time through using the flow cell. Due to this, sequencing is more effective (i.e. produces a higher yield) when a flow cell is fresh, so you will get a much higher yield from 8 samples multiplexed, than from 8 samples washed and run one after the other. There's also a bit of a risk when washing a flow cell of damaging the sequencing membrane.

[that's one of ONT's protected technologies - a polymer membrane that is transport-stable, with nice electrical properties that make it easy to load single nanopores and hard to load more than one.]

Quote:
With single flow cells being $900 (plus reagents at several hundred bucks) the cost will be more like $300 per sample, especially when you take into account that they have a limited (12 week) shelf life.
It depends on the application. A flongle flow cell with a rapid barcoding kit requiring low yield (50-100 Mb / sample) works out to about $9 / sample [as I've already mentioned]. A single whole genome sample (5-15 Gb) with a MinION flow cell and 1/6th of a ligation kit is $1000 USD for ONT reagents, plus another $100ish for NEB reagents and Ampure beads.

The 12-week shelf life is ONT's warranty period. Many ONT community users (including me) have found that flow cells will last for over a year. I have done washes of used 6-month-old flow cells with well-expired rapid kits, and got enough sequence out of the flow cells to assemble viral genomes.

For some people, I appreciate that the cost of a $100 flow cell will be beyond them, and they may not be able to take 1/144th of a rapid barcoding kit from someone who has some spare. But saying that's completely out of the price range of a hobby / home user seems a bit odd to me.
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