Originally posted by Haiqu
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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.
- Add 2.5μl fragmentation mix to 7.5μl sample in a sample tube
- Heat to 30°C for 1 min, then 80°C for 1 min
- Add 1μl adapter mix, Wait 5 mins
- Mix with sequencing buffer and load onto flow cell
Here's the barcoded sample prep:
- Add 2.5μl barcoded fragmentation mix to 7.5μl of each sample in a sample tube
- Heat to 30°C for 1 min, then 80°C for 1 min
- Mix all the prepared samples together
- Put 10μl into a new tube
- Add 1μl adapter mix, Wait 5 mins
- Mix with sequencing buffer and load onto flow cell
Sure, it's two more steps, but it doesn't involve any more learning.
A more likely scenario is use and rewash of the flow cell, where maybe 4 or 5 samples can be read at best.
It works. Depending on how much yield you want, a flow cell can be washed and re-used for 8 runs.
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]
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.
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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