Sequencing
 |
This is a note! It contains the early rough scribblings of an article, which you could grow to a more thorough page, or simply deranged science musings! ❤ |
Genome sequencing is getting more affordable. The Nanopore MinION costs USD $1000 for the Basic edition, or USD $4999 for the Enhanced edition. Which is shockingly cheap, considering what it does.. sequence DNA or RNA up to 10Gbp-20Gbp (that's giga-base-pair.)
This is shockingly cheap. There are major shortfalls of this system, but it's the only thing we can afford, and I want to get in the game now.
Background on the technology
Here's an elegant explanation of nanopore sequencers, in a delightful narrative about the history of such devices. I paraphrase:
The dominant player in DNA sequencing right now is Illumina. 23andme and Ancestry use Illumina's chips to sequence SNPs. Full, $100k Illumina sequencers use fluorescent-tagged microbeads to recognize a 150bp window of DNA, diced shotgun-style by nucleases. This window is tiny, and sequencing large genomes gets very expensive.
Nanopores are an alternative design. PCR-amplified DNA or RNA are circulated across a flow cell. Upon the cell's surface, a membrane with ion nanopores are embedded. Nucleic acids can unlock the nanopore, causing ions to pass across, and the polarity of the membrane to change. Patch clamps, tiny wires clamped on either side of the membrane, measure the voltage change.
Nanopores are (probably, too lazy to read the actual paper) laid out sequentially on each channel, in (say) ACGT order. By measuring the voltage changes in each membrane as DNA is pumped across, you read off crazy-long contiguous DNA sequences, up to 50kb+.
Nanopore is very emerging tech. These are the early days of a new generation of DNA sequencer, using a radically different architecture.
Limitations and spirited argument
Note: this is the Mk18 version. BP error rates and DOA kits seem to have improved.
This Hacker News thread has a spirited, very technical debate about the kit. At a glance:
Pros
- Cheap for what it is.
- Long, contiguous reads.
Cons
- Error rates between 10-25% per base, or according to a more recent source, 2-13%. (vs. 1% on Illuminati chips.)
- Reagents are proprietary (I think), nothing we can brew ourselves.
Maybe?
- Unclear how many samples can be analyzed on a single nanopore flow cell, before it must be chucked and a replacement acquired.
- Replacement cost per run unclear, need to investigate this.
Sanger Sequencing
- Check out this Sanger Sequencing animation.
Requirements
(Taken from here.)
Hardware
| Name | Description | |
 |
Microcentrifuge | Separates layers in a tube by density. |
 |
Vortex mixer | You use it to quickly mix tubes, just by pressing it down on the spinning head. |
 |
Thermal cycler (PCR machine) | Alternates between 30C and 80C, which makes Taq do the PCR thing. |
 |
Ice maker | You'll need an ice bucket + ice, so it's expedient to have one. |
 |
Benchtop autoclave | Simple, reliable device for autoclaving your reusable consumables! |
| Name | Description |
| OS | Windows, Mac or Ubuntu. You'll have limited tech support for Ubuntu though, be warned! |
| CPU | 4-core i7, or Xeon (don't buy Xeon, Xeon is for chumps.) Can AMD work? I'll find out.. |
| RAM | 16GB+ |
| Storage | 1TB SSD. You must use SSD! Spinning drives can't save that firehose of data in time, and it'll overwhelm the buffer in RAM. |
| USB | USB 3.0, or USB Type-C. |
| Image | Name | Description |
 |
Tube marker | You need a special kind of marker so the labels on tubes won't smudge. |
 |
Transfer pipette (micropipette) | You can dial in the precise volume to transfer between tubes. |
Consumables (stuff you need to keep buying)
- Autoclavable pipette tips, media slides, PCR tubes and containers.
- Ensure that PCR can be autoclaved.
- The MinION flow cells cost $500 each, and can apparently read 10-20Gbp each.