Charting a course to hands-on DNA sequencing with the Oxford Nanopore MinION
Like any self-respecting DIYBio enthusiast, I've been flirting with the idea of doing some hands-on sequencing, on and off for the past few years.
There were musings about doing some good old fashioned Sanger sequencing (still on the table as a workshop-type thing, in the same spirit that motivates the hand papermaking workshops you sometimes find at industry & technology museums). Then I've been eyeing the somewhat affordable but fairly outdated DNA sequencers that occasionally pop up on eBay. So far, I've always backed off for a number of reasons. Short version: It seemed impractical. They're usually still quite expensive. They're too big for my tiny biolab, so I'd have to find another place to put them. I'd have to learn how to operate them without being able to count on the manufacturer's support. I never investigated this closely, but I'm not sure that manufacturers are particularly keen on providing training to a second-hand buyer of an already paid-for machine. The same uncertainty applied to maintenance and repairs. The reagents required by the machine are also not exactly cheap. I've never enjoyed tinkering with vintage computers just for the sake of it, and the same is true for vintage sequencers.
Then, around 2014, Oxford Nanopore appeared on the scene with their MinION sequencers. They're sequencers the size and shape of a bulky USB flash drive, and they're available at a price (~1000 USD) that is still expensive if you're considering paying for one out of your own pocket, but definitely within reach. Hey, don't judge me - I hear people spend that much on a new iPhone or iPad, so there.
A few weeks ago, yet another round of "Maybe I should get a MinION" caused me to start investigating this idea in earnest. These were my main objectives:
1) Get as clear as possible a picture of what the process from raw sample to, say, a FASTQ file would look like.
2) Get an idea of the cost associated with actually using the MinION. Because, no, the $1000 will not be the end of the investment. There are consumables involved, some of which are pricey.
2.1) Can I substitute some of the recommended consumables with cheaper alternatives?
3) Figure out whether my lab is set up to perform sequencing experiments with a MinION. What equipment is needed, do I already have it, can I get it somewhere or build it? Again, there are some pieces of equipment that are needed beyond the MinION itself, though not that many; see below.
4) Figure out whether my project makes sense and the MinION would be a suitable tool: I've been wanting to explore the distribution of antibiotic resistance markers in different environments, some of them very remote. To get a good idea of whether the MinION would be a good fit for this, I need to come up with a much more detailed project plan.
Maybe there should be another objective on this list, centering around the bioinformatics side of working with MinION data. My justification for leaving it out for now: since my background is in CS and I've worked with bioinformatics software in general and sequencing data in particular (SOLiD and Illumina) before, I'm (over?)confident that I'll probably be able to figure it out.
I am glad to report that in the course of this investigation I progressed from "total confusion" to "mild confusion". Here's what I've learned.
The $1000 I mentioned above are for a MinION starter pack - it includes the MinION itself, 2 flow cells, 1 sequencing kit, and 1 wash kit. And that means?
The MinION itself is pretty much useless without the flow cells. They can be reused, but not indefinitely. After they've run for a certain time, they have to be replaced. And they have a very limited shelf life - ONT recommends using them within eight weeks of delivery.
The sequencing kit is used for library preparation - it's what you do to your sample to make it ready for MinION sequencing.
The wash kit is used to clean a flow cell between runs so you can reuse it.
So you've run some experiments, and the consumables in the starter pack are used up - what next? The cost for replacing consumables is currently as follows:
ONT provides a list of IT requirements to use the MinION.
IT-wise, I'd need:
Depending on what kind of sequencing experiment you want to run, the different sequencing kits will be more or less suitable. For example, the 2D reads generally have higher accuracy than the 1D reads. So if you're looking to catalogue single nucleotide variants, you'll probably get better results with the 2D kit. On the other hand, the 2D kit has the most extensive requirements in terms of third-party consumables. The Rapid Sequencing Kit, on the other hand, has minimal requirements for 3rd party consumables and a very short preparation time. I haven't been able to find a lot of data on the accuracy of this kit, though?
The Rapid Sequencing Kit only requires nuclease free water, pipette tips, 0.2ml PCR tubes and NEB Blunt/TA Ligase Master Mix. I already have everything on this list except the Ligase Master Mix, which costs 99 Euros for 50 reactions. That seems acceptable. I've read threads on SeqAnswers where people report using just any old ligase they had lying around, and still getting results, but since I don't have any ligase lying around and in the interest of not introducing too much variation on the "official" protocols before I get a feeling for working with the MinION, I'd probably stick with the NEB Blunt/TA Ligase Master Mix, at least at first.
The 2D Ligation Sequencing Kit, on the other hand, has a few more requirements:
I'm going to proceed cautiously by building a Hula mixer and clearing up some of the open questions I still have - cheaper alternatives for some of the consumables, working with magnetic beads, antibiotics resistance marker tracking through sequencing, to be precise. Next up would be hunting down the third-party consumables, and then finally, ordering a MinION starter pack.
If you're still with me at this point, I'd like to hear from you. Questions? Comments? Have an idea for a possible sequencing project? Interested in eventually sharing the cost for sequencing kits or bulk orders of flow cells in and around Berlin?
There were musings about doing some good old fashioned Sanger sequencing (still on the table as a workshop-type thing, in the same spirit that motivates the hand papermaking workshops you sometimes find at industry & technology museums). Then I've been eyeing the somewhat affordable but fairly outdated DNA sequencers that occasionally pop up on eBay. So far, I've always backed off for a number of reasons. Short version: It seemed impractical. They're usually still quite expensive. They're too big for my tiny biolab, so I'd have to find another place to put them. I'd have to learn how to operate them without being able to count on the manufacturer's support. I never investigated this closely, but I'm not sure that manufacturers are particularly keen on providing training to a second-hand buyer of an already paid-for machine. The same uncertainty applied to maintenance and repairs. The reagents required by the machine are also not exactly cheap. I've never enjoyed tinkering with vintage computers just for the sake of it, and the same is true for vintage sequencers.
Then, around 2014, Oxford Nanopore appeared on the scene with their MinION sequencers. They're sequencers the size and shape of a bulky USB flash drive, and they're available at a price (~1000 USD) that is still expensive if you're considering paying for one out of your own pocket, but definitely within reach. Hey, don't judge me - I hear people spend that much on a new iPhone or iPad, so there.
A few weeks ago, yet another round of "Maybe I should get a MinION" caused me to start investigating this idea in earnest. These were my main objectives:
1) Get as clear as possible a picture of what the process from raw sample to, say, a FASTQ file would look like.
2) Get an idea of the cost associated with actually using the MinION. Because, no, the $1000 will not be the end of the investment. There are consumables involved, some of which are pricey.
2.1) Can I substitute some of the recommended consumables with cheaper alternatives?
3) Figure out whether my lab is set up to perform sequencing experiments with a MinION. What equipment is needed, do I already have it, can I get it somewhere or build it? Again, there are some pieces of equipment that are needed beyond the MinION itself, though not that many; see below.
4) Figure out whether my project makes sense and the MinION would be a suitable tool: I've been wanting to explore the distribution of antibiotic resistance markers in different environments, some of them very remote. To get a good idea of whether the MinION would be a good fit for this, I need to come up with a much more detailed project plan.
Maybe there should be another objective on this list, centering around the bioinformatics side of working with MinION data. My justification for leaving it out for now: since my background is in CS and I've worked with bioinformatics software in general and sequencing data in particular (SOLiD and Illumina) before, I'm (over?)confident that I'll probably be able to figure it out.
I am glad to report that in the course of this investigation I progressed from "total confusion" to "mild confusion". Here's what I've learned.
The MinION starter pack
The $1000 I mentioned above are for a MinION starter pack - it includes the MinION itself, 2 flow cells, 1 sequencing kit, and 1 wash kit. And that means?
The MinION itself is pretty much useless without the flow cells. They can be reused, but not indefinitely. After they've run for a certain time, they have to be replaced. And they have a very limited shelf life - ONT recommends using them within eight weeks of delivery.
The sequencing kit is used for library preparation - it's what you do to your sample to make it ready for MinION sequencing.
The wash kit is used to clean a flow cell between runs so you can reuse it.
Beyond the MinION starter pack
So you've run some experiments, and the consumables in the starter pack are used up - what next? The cost for replacing consumables is currently as follows:
- Flow cell: $900 (Note: If you order 48 flow cells, the cost for each is $500. What? Why? That sucks. Regional collective flow cell orders, anyone?)
- Sequencing kit: Between $510 and $599 per kit, which includes enough reagents for six library preparations. Let's say that's shorthand for "experiments".
- Wash kit: $189; includes enough reagents for 12 washes.
IT requirements
ONT provides a list of IT requirements to use the MinION.
IT-wise, I'd need:
- Windows, macOS, or Linux are supported
- 16 GB RAM
- i7 or Xeon, >= 4 cores
- 1 TB SSDB
- USB3 port
- Windows/Linux dual boot
- 16 GB RAM
- i7-6600U with 4 cores
- 1 TB SSD - not quite. I have a 500 GB SSD that is split about 150/350 between Windows and Linux. Hmmm. Ah well, I can probably borrow a machine with these specs.
- USB3 port, yep.
Lab equipment
The equipment requirements are mostly the same for all sequencing kits (see https://store.nanoporetech.com/kits-163.html for an overview of the available sequencing kits):- Hula mixer. Don't have one, but it looks like a fun weekend Arduino project.
- Microfuge. Got it, both an old Eppendorf 5415 benchtop centrifuge and one of those tiny palm-sized centrifuges.
- Vortex mixer. Got one.
- Thermal cycler. Got one.
- Micropipettes from 1000ul down to 2ul. Got some.
- Ice bucket with ice. This one might be tricky. I used to have some ice, but I'll have to check whether it's still good.
- Pre-chilled freezer block at -20C for 200ul tubes. Tray with crushed ice and some cooling packs should work.
- Magnetic rack for magnetic bead separation - I'm not entirely sure whether there's anything special about the magnets used in magnetic bead separation, but I'm hoping not so I can build a magnetic rack? Needs further research.
- Heating block at 37C that takes 1.5 ml tubes
Consumables
The consumables differ quite a bit between different sequencing kits, and so does the cost. As far as I understand, the sequencing kits differ in three major parameters: preparation time, mode (production of 1D vs 2D reads), type of input material.Depending on what kind of sequencing experiment you want to run, the different sequencing kits will be more or less suitable. For example, the 2D reads generally have higher accuracy than the 1D reads. So if you're looking to catalogue single nucleotide variants, you'll probably get better results with the 2D kit. On the other hand, the 2D kit has the most extensive requirements in terms of third-party consumables. The Rapid Sequencing Kit, on the other hand, has minimal requirements for 3rd party consumables and a very short preparation time. I haven't been able to find a lot of data on the accuracy of this kit, though?
The Rapid Sequencing Kit only requires nuclease free water, pipette tips, 0.2ml PCR tubes and NEB Blunt/TA Ligase Master Mix. I already have everything on this list except the Ligase Master Mix, which costs 99 Euros for 50 reactions. That seems acceptable. I've read threads on SeqAnswers where people report using just any old ligase they had lying around, and still getting results, but since I don't have any ligase lying around and in the interest of not introducing too much variation on the "official" protocols before I get a feeling for working with the MinION, I'd probably stick with the NEB Blunt/TA Ligase Master Mix, at least at first.
The 2D Ligation Sequencing Kit, on the other hand, has a few more requirements:
- Agencourt AMPure XP beads. 5ml @ 314 Euros. Ooph. I've come across a paper that reports using HighPrep PCR beads instead (https://www.ncbi.nlm.nih.gov/pubmed/26840485), which is available at 50 Euros for 5ml, so I'd probably go with that.
- NEBNext End repair / dA-tailing Module. 24 reactions @ 250 USD. Haven't found a cheaper alternative yet.
- NEB Blunt/TA Ligase Master Mix. See above; 50 reactions @ 99 Euros.
- MyOne C1 Strepatividin beads. 2ml @ 497 USD. Might be able to find a cheaper substitute, but that needs more poring over datasheets.
- Covaris g-TUBE. 10 pieces @ 275 USD. Used to shear the DNA into fragments of about 6-8kbp. I read threads on SeqAnswers that say you can shear the DNA to a suitable sizes using syringes instead.
- 1.5ml DNA LoBind Eppendorf tubes. 250 pieces @ 37 Euros.
- nuclease free water, pipette tips, 0.2ml PCR tubes, 10 mM Tris-HCl pH 8.5, 70% ethanol in nuclease free water
Storage
Most of the 3rd party reagents have to be stored at -20C, the MinION flow cells need to be stored at 4-8C. I have a lab freezer and fridge, so that's all good for localized work. These requirements could prove interesting for road trips, though.Summary
So. I'm looking good on the equipment front, but the 3rd party consumables would require quite an initial investment in addition to the MinION starter kit itself. On top of that, both the sequencing kits and the flow cells come at a substantial cost. Should be a good additional encouragement to really think through any prospective sequencing experiment. On the other hand, I'd really love to carry this hands-on sequencing into the local DIYBio and hacker communities, because I'm incredibly curious about the kinds of ideas they come up with once they've seen what the technology can (and cannot) do.I'm going to proceed cautiously by building a Hula mixer and clearing up some of the open questions I still have - cheaper alternatives for some of the consumables, working with magnetic beads, antibiotics resistance marker tracking through sequencing, to be precise. Next up would be hunting down the third-party consumables, and then finally, ordering a MinION starter pack.
If you're still with me at this point, I'd like to hear from you. Questions? Comments? Have an idea for a possible sequencing project? Interested in eventually sharing the cost for sequencing kits or bulk orders of flow cells in and around Berlin?
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