<p>My bet is that the nanopore folks have (of course!) done a ton of optimisation on two key things:</p>

<ol>
<li>The DNA cleanup</li>
<li>The library preps</li>
</ol>

<p>I think that for most DNA extractions cleanups can vary a lot, but something like a Blue Pippin will do an exceptional job if you have access to one and can use it (they won't work for SUPER long DNA fragments which can't move through a gel of course). The Blue Pippin also does your size selection, so you don't burn out pores sequencing lots of little stuff. This should help yield too.</p>

<p>Then it's all down to the library prep, and @gringer's answer has some good tips on that. </p>

CC BY-SA 3.0
2017-06-10T03:52:26.343Z
2017-06-10T05:01:59.620Z
2017-06-10T05:01:59.620Z
roblanf
<p>I am working with a fungus and am using CTAB method for DNA extraction and I used rapid kit for my initial library prep but it did not work and since than I have been using ligation kit and it has been giving me very good results. I managed to get data ranging from 4GB to 13GB.</p>

CC BY-SA 4.0
2018-10-26T20:28:34.207Z
2018-10-26T20:28:34.207Z
Saidi Rahina Hussain
<p>I attended a talk by Josh Quick at PoreCampAU 2017, in which he discussed some common barriers to getting both good sequencing yield and long read length. It mostly boils down to being more careful with the sample preparation. Bear in mind that the MinION will still sequence a dirty sample, it will just be at a reduced yield. Here are my notes from that talk:</p>
<ul>
<li>The hardest thing about MinION sequencing is getting the sample in the first place</li>
<li>There are lots of different sample types and extraction methods</li>
<li>You can't get longer reads than what you put in; shit in leads to shit out</li>
<li>DNA is very stable when not moving, but very sensitive to lateral damage</li>
<li>The phenol chloroform method of DNA extraction is very good, and can be used with a phase-locked gel to make extraction easier</li>
<li>A simple salt + alcohol extraction might be the best method for extraction (because it involves the least amount of work on the DNA)</li>
<li>EDTA (e.g. as found in TE buffer, and many extraction kits) is not compatible with the rapid kit</li>
<li>The most consistently good Nanopore runs produced by Josh's lab were 1D ligations runs on R9.4; the best overall run was a phenol-chloroform extraction + rapid kit</li>
<li>John Tyson can tune himself out of a low-yield hole (via software)</li>
<li>Getting small numbers of short reads is very important</li>
<li>Suggested (and mostly untested) purification techniques: spin column (60-100kb); ethanol extraction (100-150kb), dialysis (150-250kb); low melting-point agarose plug (~1Mb, DNA extraction in situ)</li>
<li>The nanopore protocol input is in nanograms, but should really be stated as molarity; the kit expects about 0.2 pmol input</li>
<li>Picture molecules tethered to the surface of the membrane. You can then see that the flow cell density is independent of the sequence length</li>
<li>Tapestation, Qubit and Nanodrop are all a good idea; a DNA sample that can pass all three tests will work well: no short-length shoulders by Tapestation, sufficient DNA by Qubit, high purity by Nanodrop</li>
<li>RNA can interfere with sequencing; digesting RNA is recommended</li>
<li>Freezing DNA is a really bad idea. The ice crystals are very good at chopping DNA up into small pieces</li>
<li>DNA that is kept in the fridge is remarkably stable; can be kept for over two years (and probably indefinitely)</li>
</ul>
<p>For us, our best cDNA sequencing run on MinION produced over 15M reads in June 2020. We've since shifted over to doing cDNA sequencing on PromethION flow cell (using a P2 Solo), and our best so far with that run was 89M reads.</p>

CC BY-SA 4.0
2017-05-31T10:02:35.193Z
2024-07-08T07:03:57.197Z
2024-07-08T07:03:57.197Z
gringer
<p>This is a frequently-asked question within the nanopore community. Oxford Nanopore currently claims that they are able to generate run yields of 10-15 gigabases (e.g. see <a href="https://twitter.com/Clive_G_Brown/status/865211116208697344" rel="noreferrer">here</a> and <a href="https://twitter.com/Clive_G_Brown/status/860106072081739778" rel="noreferrer">here</a>), and yet it's more common to see users only managing in the 1-5 gigabase range.</p>

<p>So why the big difference in yield?</p>

<fastq><minion>
How can I improve the yield of MinION sequencing runs?
# How can I improve the yield of MinION sequencing runs?

(Asked by: username_0 on 2017-05-31)

This is a frequently-asked question within the nanopore community. Oxford Nanopore currently claims that they are able to generate run yields of 10-15 gigabases (e.g. see [here](https://twitter.com/Clive_G_Brown/status/865211116208697344) and [here](https://twitter.com/Clive_G_Brown/status/860106072081739778)), and yet it's more common to see users only managing in the 1-5 gigabase range.

So why the big difference in yield?

**Tags:** fastq, minion

---

## 3 answers

### Answer by username_0 on 2017-05-31. Score: 14 | Accepted answer

I attended a talk by Josh Quick at PoreCampAU 2017, in which he discussed some common barriers to getting both good sequencing yield and long read length. It mostly boils down to being more careful with the sample preparation. Bear in mind that the MinION will still sequence a dirty sample, it will just be at a reduced yield. Here are my notes from that talk:

* The hardest thing about MinION sequencing is getting the sample in the first place
* There are lots of different sample types and extraction methods
* You can't get longer reads than what you put in; shit in leads to shit out
* DNA is very stable when not moving, but very sensitive to lateral damage
* The phenol chloroform method of DNA extraction is very good, and can be used with a phase-locked gel to make extraction easier
* A simple salt + alcohol extraction might be the best method for extraction (because it involves the least amount of work on the DNA)
* EDTA (e.g. as found in TE buffer, and many extraction kits) is not compatible with the rapid kit
* The most consistently good Nanopore runs produced by Josh's lab were 1D ligations runs on R9.4; the best overall run was a phenol-chloroform extraction + rapid kit
* John Tyson can tune himself out of a low-yield hole (via software)
* Getting small numbers of short reads is very important
* Suggested (and mostly untested) purification techniques: spin column (60-100kb); ethanol extraction (100-150kb), dialysis (150-250kb); low melting-point agarose plug (~1Mb, DNA extraction in situ)
* The nanopore protocol input is in nanograms, but should really be stated as molarity; the kit expects about 0.2 pmol input
* Picture molecules tethered to the surface of the membrane. You can then see that the flow cell density is independent of the sequence length
* Tapestation, Qubit and Nanodrop are all a good idea; a DNA sample that can pass all three tests will work well: no short-length shoulders by Tapestation, sufficient DNA by Qubit, high purity by Nanodrop
* RNA can interfere with sequencing; digesting RNA is recommended
* Freezing DNA is a really bad idea. The ice crystals are very good at chopping DNA up into small pieces
* DNA that is kept in the fridge is remarkably stable; can be kept for over two years (and probably indefinitely)

For us, our best cDNA sequencing run on MinION produced over 15M reads in June 2020. We've since shifted over to doing cDNA sequencing on PromethION flow cell (using a P2 Solo), and our best so far with that run was 89M reads.


---

### Answer by username_1 on 2017-06-10. Score: 2

My bet is that the nanopore folks have (of course!) done a ton of optimisation on two key things:

1. The DNA cleanup
2. The library preps

I think that for most DNA extractions cleanups can vary a lot, but something like a Blue Pippin will do an exceptional job if you have access to one and can use it (they won't work for SUPER long DNA fragments which can't move through a gel of course). The Blue Pippin also does your size selection, so you don't burn out pores sequencing lots of little stuff. This should help yield too.

Then it's all down to the library prep, and @gringer's answer has some good tips on that.


---

### Answer by username_2 on 2018-10-26. Score: 1

I am working with a fungus and am using CTAB method for DNA extraction and I used rapid kit for my initial library prep but it did not work and since than I have been using ligation kit and it has been giving me very good results. I managed to get data ranging from 4GB to 13GB.


---

