4 ms·

Do you think something like the Oxford Nanopore MinION [0] is a viable alternative, assuming you'd want to do just the sequencing yourself? I suspect most peopl

Do you think something like the Oxford Nanopore MinION [0] is a viable alternative, assuming you'd want to do just the sequencing yourself? I suspect most people wouldn't be able to do the prep without proper wet lab training and equipment [1], but the starter kit is $1000 for the device, a single flow cell, and one rapid sequencing kit (SQK-RAD004). [0]: https://nanoporetech.com/products/minion https://nanoporetech.com/products/minion [1]: https://www.youtube.com/watch?v=iS1pz3IhJvU https://www.youtube.com/watch?v=iS1pz3IhJvU

aroch 4y ago
Not for home gamers, no. ONT is fairly low throughput and the prep is surprisingly difficult to do well without experience. You would need to run tens of flow cells to get enough sequencing data.
- mbreese 4y ago
  Not to mention there is a good chunk of required "extra" equipment that is expected (centrifuges, etc) that bring the initial cost to well over $1000. You could buy most thing used, but some of the reagents could be difficult to acquire outside of a traditional lab.
  - ejstronge 4y ago
    This is not strictly true - there is a transposase based prep that does not require special equipment. Still, ONT users would need to understand how to convert raw reads into useable basecalls at loci of clinical interest
    - mbreese 4y ago
      Does it still require a pipettor, or could it work with just a transfer pipet? Their field kit should probably be as minimal as it could be. Last I looked into it, it was a minimal set of extra equipment -- votexer, centrifuge, etc... nothing unusual for a bio lab. And even most of that could be handled with a good snap of the wrist, if you weren't trying to be super precise.
      - ejstronge 4y ago
        If one is patient, the only tool needed in the wet lab is a transfer pipettor and a scale - I think this could be done without a micropipettor
xipho 4y ago
I suspect many people that frequent HN could use the MinION to generate the raw data, but generating gigabytes of DNA reads != assembling a genome. Remember, only this year the very first genome for any human was "completed". You're going to get thousands of overlapping reads, of varying lengths. Then you'll need serious processing power to combine these overlaps, then overlap the overlaps, so to speak, and on and on. What software to pick, how to parameterize/use it, this is the job of post-docs and others who like to tear their hair out. I haven't looked lately, but many one-off scientific machines have their own propitiatory binary versions of the data, for vendor lock in, so that you must also buy their crappy software to process it, double check that this isn't the case. When your first run fails, are you willing to pay again as much for the kits to run it again (these machines are very much following the cheap printer/expensive ink model IMO)? Once you have some data, do you know how you will BLAST it against annotated genomes to figure out if you have mutation X? How do you interpret e-scores, etc? For the OP company, when they say "download the data" do they mean the raw reads, or assemblies? Make sure this is spelled out (it likely is, I haven't looked lately). Do the downloads/service provide adequate metadata on the data generation process so that you can tease out errors in reads from reality (real single-nucleotide mutations), etc.? All fun stuff, but only for a very serious hobbyist, so to speak.
- aroch 4y ago
  No one should be BLASTing individual reads... And pretty much no one is going to be assembling with OLC, even for ONT data. On the products page it says they provide FASTQs, aligned BAM and VCF. Which is exactly what one would expect. They're almost certainly just running the DRAGEN pipeline (or something similar) and giving you the output it creates.
- jefftk 4y ago
  I think your typical programmer could automate the alignment, even without reading about existing algorithms. It isn't much harder than a standard interview question, especially if you're willing to use your hardware inefficiently? You aren't trying to sequence the human genome from nothing, but instead have a reference genome to work with. You take each of the reads you get from the sequencer and find where it best matches up with the reference genome. I think the wet portion is a far bigger challenge for the typical computer programmer hobbyist. (Also I don't think $1,000 will get you, in addition to the sequencer, a flow cell sufficient to gather enough reads to tell the difference between sequencing errors and mutations?)

aroch 4y ago

Not for home gamers, no. ONT is fairly low throughput and the prep is surprisingly difficult to do well without experience. You would need to run tens of flow cells to get enough sequencing data.

mbreese 4y ago
Not to mention there is a good chunk of required "extra" equipment that is expected (centrifuges, etc) that bring the initial cost to well over $1000. You could buy most thing used, but some of the reagents could be difficult to acquire outside of a traditional lab.
- ejstronge 4y ago
  This is not strictly true - there is a transposase based prep that does not require special equipment. Still, ONT users would need to understand how to convert raw reads into useable basecalls at loci of clinical interest
  - mbreese 4y ago
    Does it still require a pipettor, or could it work with just a transfer pipet? Their field kit should probably be as minimal as it could be. Last I looked into it, it was a minimal set of extra equipment -- votexer, centrifuge, etc... nothing unusual for a bio lab. And even most of that could be handled with a good snap of the wrist, if you weren't trying to be super precise.
    - ejstronge 4y ago
      If one is patient, the only tool needed in the wet lab is a transfer pipettor and a scale - I think this could be done without a micropipettor

xipho 4y ago

I suspect many people that frequent HN could use the MinION to generate the raw data, but generating gigabytes of DNA reads != assembling a genome. Remember, only this year the very first genome for any human was "completed". You're going to get thousands of overlapping reads, of varying lengths. Then you'll need serious processing power to combine these overlaps, then overlap the overlaps, so to speak, and on and on. What software to pick, how to parameterize/use it, this is the job of post-docs and others who like to tear their hair out. I haven't looked lately, but many one-off scientific machines have their own propitiatory binary versions of the data, for vendor lock in, so that you must also buy their crappy software to process it, double check that this isn't the case. When your first run fails, are you willing to pay again as much for the kits to run it again (these machines are very much following the cheap printer/expensive ink model IMO)? Once you have some data, do you know how you will BLAST it against annotated genomes to figure out if you have mutation X? How do you interpret e-scores, etc? For the OP company, when they say "download the data" do they mean the raw reads, or assemblies? Make sure this is spelled out (it likely is, I haven't looked lately). Do the downloads/service provide adequate metadata on the data generation process so that you can tease out errors in reads from reality (real single-nucleotide mutations), etc.? All fun stuff, but only for a very serious hobbyist, so to speak.

aroch 4y ago
No one should be BLASTing individual reads... And pretty much no one is going to be assembling with OLC, even for ONT data. On the products page it says they provide FASTQs, aligned BAM and VCF. Which is exactly what one would expect. They're almost certainly just running the DRAGEN pipeline (or something similar) and giving you the output it creates.
jefftk 4y ago
I think your typical programmer could automate the alignment, even without reading about existing algorithms. It isn't much harder than a standard interview question, especially if you're willing to use your hardware inefficiently? You aren't trying to sequence the human genome from nothing, but instead have a reference genome to work with. You take each of the reads you get from the sequencer and find where it best matches up with the reference genome. I think the wet portion is a far bigger challenge for the typical computer programmer hobbyist. (Also I don't think $1,000 will get you, in addition to the sequencer, a flow cell sufficient to gather enough reads to tell the difference between sequencing errors and mutations?)