5 ms·

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, o

by 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?)
- mbreese 4y ago
  > your typical programmer could automate the assembly Alignment. You could easily automate alignment of raw sequencing reads to their position in the reference genome. From there, even variant calling (finding your individual changes from the reference) is pretty easy as well. Assembly is a bigger challenge that takes the raw reads and puts them together into a larger (graph) structure. This is usually de-novo, but could be able to be done with a reference as well. But this is not really something that is routine or possible with 30X sequence coverage to the degree one would expect. The wet-lab portion isn't terrible and anyone with skills making anything (soldering, for example), should be able to do it -- once you figured out some of the jargon. The bigger challenge someone not trained in this would have is interpreting the variations that you'd find. This can be quite difficult to figure out if an A>C at a particular position is meaningful or not. Again, much of these can be filtered using existing tools, but even then, it can be quite difficult to interpret results.
  - jefftk 4y ago
    > Alignment Whoops; fixed! > This can be quite difficult to figure out if an A>C at a particular position is meaningful or not. What about https://promethease.com https://promethease.com ?
    - mbreese 4y ago
      Promethease isn’t a bad place to start. I don’t know how it works with uncharacterized variants, but it would be a good place to start. Note: I do this work for cancer genomes (WGS, tumor and germline). Promethease is not something that I use for annotations, but my workflows are significantly more complex. The biggest thing I’d look out for is mindset. Biology is very different from comp sci. CS is very deterministic. You change something here, you get a result there. Biology, on the other hand, is very messy. There are compensation mechanisms on top of compensation mechanisms, and everything favors life. If you see a deleterious variant, that still doesn’t mean you’ll see a phenotype (effect on you). It’s quite a different world and can be very scary if you are looking at your own data.

by xipho 4y ago

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?)

mbreese 4y ago
> your typical programmer could automate the assembly Alignment. You could easily automate alignment of raw sequencing reads to their position in the reference genome. From there, even variant calling (finding your individual changes from the reference) is pretty easy as well. Assembly is a bigger challenge that takes the raw reads and puts them together into a larger (graph) structure. This is usually de-novo, but could be able to be done with a reference as well. But this is not really something that is routine or possible with 30X sequence coverage to the degree one would expect. The wet-lab portion isn't terrible and anyone with skills making anything (soldering, for example), should be able to do it -- once you figured out some of the jargon. The bigger challenge someone not trained in this would have is interpreting the variations that you'd find. This can be quite difficult to figure out if an A>C at a particular position is meaningful or not. Again, much of these can be filtered using existing tools, but even then, it can be quite difficult to interpret results.
- jefftk 4y ago
  > Alignment Whoops; fixed! > This can be quite difficult to figure out if an A>C at a particular position is meaningful or not. What about https://promethease.com https://promethease.com ?
  - mbreese 4y ago
    Promethease isn’t a bad place to start. I don’t know how it works with uncharacterized variants, but it would be a good place to start. Note: I do this work for cancer genomes (WGS, tumor and germline). Promethease is not something that I use for annotations, but my workflows are significantly more complex. The biggest thing I’d look out for is mindset. Biology is very different from comp sci. CS is very deterministic. You change something here, you get a result there. Biology, on the other hand, is very messy. There are compensation mechanisms on top of compensation mechanisms, and everything favors life. If you see a deleterious variant, that still doesn’t mean you’ll see a phenotype (effect on you). It’s quite a different world and can be very scary if you are looking at your own data.