4 ms·

Indel mutation rates aren't high enough, and the number of cells screened for mutations is too low for this explanation to suffice.

nonbel 8y ago
Do you have a paper in mind? From what I've seen 0.01% - 10% of controls have indels at any given site (obviously this depends on treatment, cell type, etc). You could call that noise of course, at the very least they can't detect rates below those using current methods. I'll go find some refs on it if you ask.
- azeotropic 8y ago
  You must be misunderstanding what you've read. There's no way cells survive with indels at 10% of their base positions, that's not possible.
  - nonbel 8y ago
    Thats not what I said. I said some significant percent of cells (I remember as high as 10% from one paper, but usually its closer to 0.1%...) seem to have indels at any given locus. Either that or the methods used to measure this are too noisy to detect mutants that exist at those rates.
    - azeotropic 8y ago
      How is the per-base indel rate not 10% if 10% of cells carry an indel at any given locus?
      - nonbel 8y ago
        Reference: GAATTC Variants: Cell 1: TAATTC Cell 2: GTATTC Cell 3: GATTTC Cell 4: GAAGTC Cell 5: GAATGC Cell 6: GAATTT Cell 7: GAATTC Cell 8: GAATTC 7/8 cells contain the reference nucleotide at any given site. Ie, 12.5% of cells contain an indel wherever you look. For six cells, 1/6 nucleotides differ from the reference sequence (16.7% per base indel rate). For two cells, there is no difference (0 % per base indel rate). Even though 12.5% of cells contain an indel at any given site, no single cell contains 12.5% indels. There is no reason the two values should be the same.
        azeotropic 8y ago
        You clearly have no idea what you are talking about. 1) There are no indels in your example, only nucleotide substitutions. 2) There are six substitutions in 48 bases, for a per-base substitution rate of 12.5%. The fact that none of the individual cells contains 12.5% substitutions is totally irrelevant.
        nonbel 8y ago
        >"There are no indels in your example, only nucleotide substitutions." It works perfectly fine as an example. A single nucleotide change could be due to an indel: "Indel is a molecular biology term for an insertion or deletion of bases in the genome of an organism. It is classified among small genetic variations, measuring from 1 to 10 000 base pairs in length,[1][2][3][4][5][6][7]" https://en.wikipedia.org/wiki/Indel https://en.wikipedia.org/wiki/Indel >"There are six substitutions in 48 bases, for a per-base substitution rate of 12.5%. The fact that none of the individual cells contains 12.5% substitutions is totally irrelevant." In the experiments they are counting how many cells contain a mutant (eg via GFP expression or not). Thus the number you want is percent of cells that contain a mutation at a given site. You are the one calculating some other number... Remember what we are discussing? Its percent of cells with a given mutation and mutations per cell, the numbers I calculated: >"There's no way cells survive with indels at 10% of their base positions, that's not possible." >"You clearly have no idea what you are talking about." There seems to be some heavy dunning-kruger going on with your post.
        azeotropic 8y ago
        I asserted that saying that 10% of cells have an indel at any given site is equivalent to saying the per-base indel mutation rate is 10%. You posted an example purporting to disprove this, but instead, illustrated that you don't know what an indel is, and that the two statements are indeed equivalent -- at any given site, 12.5% of cells are mutant, and that the per-base mutation rate is 12.5%. The only one with Dunning-Kruger is you.
        8y ago
  - nonbel 8y ago
    Actually just look at the controls in this paper: Fig 1b: 0.31% Fig 3b: 1.12% Fig 4b: 0.18%
    - azeotropic 8y ago
      Which paper? OPs, or the one about T-cells that you posted?
      - nonbel 8y ago
        The OP paper: https://www.nature.com/articles/Nbt.4192 https://www.nature.com/articles/Nbt.4192
        azeotropic 8y ago
        Those are not direct measures of indel rates, those are measures of PigA and CD9 protein levels above the threshold set by the investigators. The main text makes it clear that those percentages should be regarded as background noise, and they adjust for it when comparing CD9 and PigA targeting.
        nonbel 8y ago
        They can't detect the presence of mutants if hey occur at frequencies at or below the background rates. I mentioned this multiple times. Its simply that no one knows if they are present or not. Earlier: >"You could call that noise of course, at the very least they can't detect rates below those using current methods." >"Either that or the methods used to measure this are too noisy to detect mutants that exist at those rates."

nonbel 8y ago

Do you have a paper in mind? From what I've seen 0.01% - 10% of controls have indels at any given site (obviously this depends on treatment, cell type, etc). You could call that noise of course, at the very least they can't detect rates below those using current methods. I'll go find some refs on it if you ask.

azeotropic 8y ago
You must be misunderstanding what you've read. There's no way cells survive with indels at 10% of their base positions, that's not possible.
- nonbel 8y ago
  Thats not what I said. I said some significant percent of cells (I remember as high as 10% from one paper, but usually its closer to 0.1%...) seem to have indels at any given locus. Either that or the methods used to measure this are too noisy to detect mutants that exist at those rates.
  - azeotropic 8y ago
    How is the per-base indel rate not 10% if 10% of cells carry an indel at any given locus?
    - nonbel 8y ago
      Reference: GAATTC Variants: Cell 1: TAATTC Cell 2: GTATTC Cell 3: GATTTC Cell 4: GAAGTC Cell 5: GAATGC Cell 6: GAATTT Cell 7: GAATTC Cell 8: GAATTC 7/8 cells contain the reference nucleotide at any given site. Ie, 12.5% of cells contain an indel wherever you look. For six cells, 1/6 nucleotides differ from the reference sequence (16.7% per base indel rate). For two cells, there is no difference (0 % per base indel rate). Even though 12.5% of cells contain an indel at any given site, no single cell contains 12.5% indels. There is no reason the two values should be the same.
      - azeotropic 8y ago
        You clearly have no idea what you are talking about. 1) There are no indels in your example, only nucleotide substitutions. 2) There are six substitutions in 48 bases, for a per-base substitution rate of 12.5%. The fact that none of the individual cells contains 12.5% substitutions is totally irrelevant.
        nonbel 8y ago
        >"There are no indels in your example, only nucleotide substitutions." It works perfectly fine as an example. A single nucleotide change could be due to an indel: "Indel is a molecular biology term for an insertion or deletion of bases in the genome of an organism. It is classified among small genetic variations, measuring from 1 to 10 000 base pairs in length,[1][2][3][4][5][6][7]" https://en.wikipedia.org/wiki/Indel https://en.wikipedia.org/wiki/Indel >"There are six substitutions in 48 bases, for a per-base substitution rate of 12.5%. The fact that none of the individual cells contains 12.5% substitutions is totally irrelevant." In the experiments they are counting how many cells contain a mutant (eg via GFP expression or not). Thus the number you want is percent of cells that contain a mutation at a given site. You are the one calculating some other number... Remember what we are discussing? Its percent of cells with a given mutation and mutations per cell, the numbers I calculated: >"There's no way cells survive with indels at 10% of their base positions, that's not possible." >"You clearly have no idea what you are talking about." There seems to be some heavy dunning-kruger going on with your post.
        azeotropic 8y ago
        I asserted that saying that 10% of cells have an indel at any given site is equivalent to saying the per-base indel mutation rate is 10%. You posted an example purporting to disprove this, but instead, illustrated that you don't know what an indel is, and that the two statements are indeed equivalent -- at any given site, 12.5% of cells are mutant, and that the per-base mutation rate is 12.5%. The only one with Dunning-Kruger is you.
        8y ago
- nonbel 8y ago
  Actually just look at the controls in this paper: Fig 1b: 0.31% Fig 3b: 1.12% Fig 4b: 0.18%
  - azeotropic 8y ago
    Which paper? OPs, or the one about T-cells that you posted?
    - nonbel 8y ago
      The OP paper: https://www.nature.com/articles/Nbt.4192 https://www.nature.com/articles/Nbt.4192
      - azeotropic 8y ago
        Those are not direct measures of indel rates, those are measures of PigA and CD9 protein levels above the threshold set by the investigators. The main text makes it clear that those percentages should be regarded as background noise, and they adjust for it when comparing CD9 and PigA targeting.
        nonbel 8y ago
        They can't detect the presence of mutants if hey occur at frequencies at or below the background rates. I mentioned this multiple times. Its simply that no one knows if they are present or not. Earlier: >"You could call that noise of course, at the very least they can't detect rates below those using current methods." >"Either that or the methods used to measure this are too noisy to detect mutants that exist at those rates."