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Was thinking the same and wondering how did they obtain the million-base long sequences. Turned out they used short read sequencing with 150 or 250 bp reads and

Was thinking the same and wondering how did they obtain the million-base long sequences. Turned out they used short read sequencing with 150 or 250 bp reads and computationally assembled the long reads [1]. While this is a traditionally valid method, the newer long read sequencing technologies such as Oxford Nanopore or PacBio would be an more appropriate and direct method. [1] https://www.biorxiv.org/content/10.1101/2021.07.10.451761v1.full.pdf https://www.biorxiv.org/content/10.1101/2021.07.10.451761v1....

f6v 5y ago
Long reads are still not as widely used since the error rates are much higher than in shotgun approach.
- tdido 5y ago
  Actually, for the use case of genome assemblies you can compensate for the error rate with depth of coverage. Long reads are already the state-of-the-art for assembling genomes and are allowing us to get information that is simply invisible using short reads. https://www.biorxiv.org/content/10.1101/2021.05.26.445798v1 https://www.biorxiv.org/content/10.1101/2021.05.26.445798v1
  - f6v 5y ago
    It's definitely getting better, but errors have to be corrected computationally, and it still seems like a big challenge.
  - londons_explore 5y ago
    One would think a hybrid approach will bring the best results...? Long reads help with alignment and arrangement, and short reads eliminate small errors of just a few base pairs.
ejstronge 5y ago
PacBio reads don't extend to the million base length and are a considerably more expensive approach than short read sequencing if assembly is happening anyway. ONT is a great tool but the goal of this report isn't necessarily to show that these molecules are 1 million bases in length vs showing that they represent novel DNA sequences. In summary, ONT and PacBio are neither more appropriate or necessarily more direct methods here.
- andrewon 5y ago
  Would have to disagree. They will need to confirm these were not artifacts. Long read sequencing assembly is the best approach to show the computationally assembled reads indeed coming from single contiguous molecules.
  - ejstronge 5y ago
    I'm not sure what kind of artifact would assemble into a series of novel ORFs but I suppose we will indeed have to disagree.

f6v 5y ago

Long reads are still not as widely used since the error rates are much higher than in shotgun approach.

tdido 5y ago
Actually, for the use case of genome assemblies you can compensate for the error rate with depth of coverage. Long reads are already the state-of-the-art for assembling genomes and are allowing us to get information that is simply invisible using short reads. https://www.biorxiv.org/content/10.1101/2021.05.26.445798v1 https://www.biorxiv.org/content/10.1101/2021.05.26.445798v1
- f6v 5y ago
  It's definitely getting better, but errors have to be corrected computationally, and it still seems like a big challenge.
- londons_explore 5y ago
  One would think a hybrid approach will bring the best results...? Long reads help with alignment and arrangement, and short reads eliminate small errors of just a few base pairs.

ejstronge 5y ago

PacBio reads don't extend to the million base length and are a considerably more expensive approach than short read sequencing if assembly is happening anyway. ONT is a great tool but the goal of this report isn't necessarily to show that these molecules are 1 million bases in length vs showing that they represent novel DNA sequences. In summary, ONT and PacBio are neither more appropriate or necessarily more direct methods here.

andrewon 5y ago
Would have to disagree. They will need to confirm these were not artifacts. Long read sequencing assembly is the best approach to show the computationally assembled reads indeed coming from single contiguous molecules.
- ejstronge 5y ago
  I'm not sure what kind of artifact would assemble into a series of novel ORFs but I suppose we will indeed have to disagree.