5 ms·

It's cool that nanopore technologies are getting this affordable, but keep in mind that these technologies (to my knowledge) still have very high error rates co

It's cool that nanopore technologies are getting this affordable, but keep in mind that these technologies (to my knowledge) still have very high error rates compared older sequencing techniques. Both in terms of individual nucleotides (A, C, G, and Ts) being misread, but also in terms of stretches of nucleotides being mistakenly added to or removed from the resulting sequences (indels). So, yes, you can sequence your genome relatively cheaply using these technologies at home, but you won't be able to draw any conclusions from the results

greazy 1y ago
With the recent R10 flow cells the error rate has improved. The basecalling models have also been steadily improving and therefore reducing the error rate. For assembling a bacterial genome the consensus error rate is as low or in some cases better than Illumina. Nanopore platform has its usecases that Illumina falls short on. > So, yes, you can sequence your genome relatively cheaply using these technologies at home, but you won't be able to draw any conclusions from the results Agreed, any at home sequencing should not be used to draw any conclusions.
Ovah 1y ago
That's a prevalent misconception even in the scientific community. Sure, each read has 1% incorrect bases (0.01). But each segment of DNA is read many times over. More or less 0.01^(many times) ≈ 0 incorrect bases.
- optionalsquid 1y ago
  The author got less than 1x coverage for their efforts. To get the kind of coverage required for reliable base-calls, you need significantly higher coverage, and therefore a significantly higher spend
- bonsai_spool 1y ago
  > That's a prevalent misconception even in the scientific community. Sure, each read has 1% incorrect bases (0.01). But each segment of DNA is read many times over. More or less 0.01^(many times) ≈ 0 incorrect bases. That's true in targeted sequencing, but when you try to sequence a whole genome, this is unlikely.
  - optionalsquid 1y ago
    > That's true in targeted sequencing, but when you try to sequence a whole genome, this is unlikely. Whole-genome shotgun sequencing is pretty cheap these days. The person you are replying to doesn't give any specific numbers, but in my experience, you aim for 5-20x average coverage for population level studies, depending on the number of samples and what you are looking for, and 30x or higher for studies where individuals are important. For context, coverage refers to the (average) number of resulting DNA sequences that cover a given position in the target genome. Though there is of course variation in local coverage, regardless of your average coverage, and that can result in individual base-calls being being more or less reliable
    - bonsai_spool 1y ago
      I’m referring to the experiment done in the OP - the most I’ve read about from an minION flow cell is 8 Gb (and this is from cell line preps with tons of DNA, so the coverage isn’t great). You need multiple flow cells or a higher capacity flow cell to get anything close to 1X on an unselected genome prep. Shotgun sequencing isn’t probably what you meant to say - this is all enzymatic or, if it’s sonicated, gets size selected.
      - optionalsquid 1y ago
        What the person you replied to described read like short read sequencing with PCR amplification to me ("each segment of DNA is read many times over"), rather than nanopore sequencing. My reply to you was written based on that (possibly false) assumption. But if we are talking nanopore sequencing, then yes, you need multiple flowcells. Which is not a problem if you are not a private person attempting to sequence your own genome on the cheap
        bonsai_spool 1y ago
        There wasn’t enough information to tell (on my 1 minute scan) which nanopore kit was used, but the presence of PCR does not imply short reads. You can do nanopore PCR/cDNA workflows right up to the largest known mRNAs (13kb). Edit: I’m not sure if you’re saying that you can’t do a 5/20/30X genome on nanopore - that’s also not true. It only makes sense in particular research settings, of course.

greazy 1y ago

With the recent R10 flow cells the error rate has improved. The basecalling models have also been steadily improving and therefore reducing the error rate. For assembling a bacterial genome the consensus error rate is as low or in some cases better than Illumina. Nanopore platform has its usecases that Illumina falls short on. > So, yes, you can sequence your genome relatively cheaply using these technologies at home, but you won't be able to draw any conclusions from the results Agreed, any at home sequencing should not be used to draw any conclusions.

Ovah 1y ago

That's a prevalent misconception even in the scientific community. Sure, each read has 1% incorrect bases (0.01). But each segment of DNA is read many times over. More or less 0.01^(many times) ≈ 0 incorrect bases.

optionalsquid 1y ago
The author got less than 1x coverage for their efforts. To get the kind of coverage required for reliable base-calls, you need significantly higher coverage, and therefore a significantly higher spend
bonsai_spool 1y ago
> That's a prevalent misconception even in the scientific community. Sure, each read has 1% incorrect bases (0.01). But each segment of DNA is read many times over. More or less 0.01^(many times) ≈ 0 incorrect bases. That's true in targeted sequencing, but when you try to sequence a whole genome, this is unlikely.
- optionalsquid 1y ago
  > That's true in targeted sequencing, but when you try to sequence a whole genome, this is unlikely. Whole-genome shotgun sequencing is pretty cheap these days. The person you are replying to doesn't give any specific numbers, but in my experience, you aim for 5-20x average coverage for population level studies, depending on the number of samples and what you are looking for, and 30x or higher for studies where individuals are important. For context, coverage refers to the (average) number of resulting DNA sequences that cover a given position in the target genome. Though there is of course variation in local coverage, regardless of your average coverage, and that can result in individual base-calls being being more or less reliable
  - bonsai_spool 1y ago
    I’m referring to the experiment done in the OP - the most I’ve read about from an minION flow cell is 8 Gb (and this is from cell line preps with tons of DNA, so the coverage isn’t great). You need multiple flow cells or a higher capacity flow cell to get anything close to 1X on an unselected genome prep. Shotgun sequencing isn’t probably what you meant to say - this is all enzymatic or, if it’s sonicated, gets size selected.
    - optionalsquid 1y ago
      What the person you replied to described read like short read sequencing with PCR amplification to me ("each segment of DNA is read many times over"), rather than nanopore sequencing. My reply to you was written based on that (possibly false) assumption. But if we are talking nanopore sequencing, then yes, you need multiple flowcells. Which is not a problem if you are not a private person attempting to sequence your own genome on the cheap
      - bonsai_spool 1y ago
        There wasn’t enough information to tell (on my 1 minute scan) which nanopore kit was used, but the presence of PCR does not imply short reads. You can do nanopore PCR/cDNA workflows right up to the largest known mRNAs (13kb). Edit: I’m not sure if you’re saying that you can’t do a 5/20/30X genome on nanopore - that’s also not true. It only makes sense in particular research settings, of course.