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> your typical programmer could automate the assembly Alignment. You could easily automate alignment of raw sequencing reads to their position in the reference

> 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.
  - jefftk 4y ago
    I think the biggest reason it's scary is a statistics literacy problem: everyone has lots of mutations, and a mutation is much more likely to be bad than good. This means that when people look at a Prometheus report and see a long list of deleterious mutations they tend to take it, overall, as bad news: so much red! But since that's what you would expect to see even before looking at the report it shouldn't move your opinion either way.
    - mbreese 4y ago
      My prior is that any random variant would likely do nothing. It’s all about location, location, location. First off, a variant would need to be in a gene to do something (not entirely true, but a good enough approximation). Then, the variant would need to be in a coding region/exon. Finally, the variant would need to change the amino acid (there is a Twitter/biorxiv war going on about this specifically now). You have approximately 0.1% unique DNA sequence compared to someone else. But there are 3 billion bp in the human genome. So that’s an expected 3 million variants. And that’s just to start. And just to add to the prior probabilities — you’re probably living, right? So, whatever variants you might find haven’t been lethal yet! (Even better if you’ve made it to adulthood!) And to complicate things further, one variant in a gene could do nothing if the other copy is still good. Even if you have two bad variants in a single gene, they might be on the same allele (from the same parent). In which case, you still have one good copy! It’s like how Iceman got hit twice in the same engine at the end of the original Top Gun. He still had one good engine to get home. For many genes, that’s all you need. For others, you might not even need one good copy as other genes could take over the same job. Yes, there are some variants that you don’t want to see. Things that might indicate high likelihood of disease down the road. But even that is bound by statistical probabilities. Good news — some of those can’t be seen by this type of sequencing. You’re absolutely right though — it can be scary. After looking at many genomes, it can be amazing that we all work as well as we do.
- topynate 4y ago
  I used https://genvue.geneticgenie.org/ https://genvue.geneticgenie.org/ – it's free and I've found it to be the best starting point for further investigation. I also have access to Nebula Genomics' tools as part of the package I paid for. Yes, there is a fear factor when you're looking at your own genome. On the other hand, seeing you have an increased risk of something can be quite motivating to make some compensating lifestyle changes.

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.
- jefftk 4y ago
  I think the biggest reason it's scary is a statistics literacy problem: everyone has lots of mutations, and a mutation is much more likely to be bad than good. This means that when people look at a Prometheus report and see a long list of deleterious mutations they tend to take it, overall, as bad news: so much red! But since that's what you would expect to see even before looking at the report it shouldn't move your opinion either way.
  - mbreese 4y ago
    My prior is that any random variant would likely do nothing. It’s all about location, location, location. First off, a variant would need to be in a gene to do something (not entirely true, but a good enough approximation). Then, the variant would need to be in a coding region/exon. Finally, the variant would need to change the amino acid (there is a Twitter/biorxiv war going on about this specifically now). You have approximately 0.1% unique DNA sequence compared to someone else. But there are 3 billion bp in the human genome. So that’s an expected 3 million variants. And that’s just to start. And just to add to the prior probabilities — you’re probably living, right? So, whatever variants you might find haven’t been lethal yet! (Even better if you’ve made it to adulthood!) And to complicate things further, one variant in a gene could do nothing if the other copy is still good. Even if you have two bad variants in a single gene, they might be on the same allele (from the same parent). In which case, you still have one good copy! It’s like how Iceman got hit twice in the same engine at the end of the original Top Gun. He still had one good engine to get home. For many genes, that’s all you need. For others, you might not even need one good copy as other genes could take over the same job. Yes, there are some variants that you don’t want to see. Things that might indicate high likelihood of disease down the road. But even that is bound by statistical probabilities. Good news — some of those can’t be seen by this type of sequencing. You’re absolutely right though — it can be scary. After looking at many genomes, it can be amazing that we all work as well as we do.
topynate 4y ago
I used https://genvue.geneticgenie.org/ https://genvue.geneticgenie.org/ – it's free and I've found it to be the best starting point for further investigation. I also have access to Nebula Genomics' tools as part of the package I paid for. Yes, there is a fear factor when you're looking at your own genome. On the other hand, seeing you have an increased risk of something can be quite motivating to make some compensating lifestyle changes.