3 ms·

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.
      - nonbel 8y ago
        Here is what I am responding to (also keep in mind I was using indel to mean "indels and substitutions", ie whatever may be counted as an error during NHEJ): > "There's no way cells survive with indels at 10% of their base positions, that's not possible." > "How is the per-base indel rate not 10% if 10% of cells carry an indel at any given locus?" I figured you were referring to per base within each cell. Your overall "per base" rate is getting the average (across cells) number of mutated sites. The survival of a cell is determined by its own genome, not the average of the population its been grouped into. However, it is the case that if x% of cells have a mutation at any given site then there must be at least some cells in this group that contain x% or greater mutated sites. Is that what you meant? Because that's not a bad point. I don't know where that 10% value came from and have no attachment to it, just assume that one is measurement noise for now, but even cells with 0.1% of sites mutated seems like a lot. There doesn't seem to be many whole genome single cell sequencing results available yet, which is what I think is needed here. Here is one that reports ~ 15k SNVs, 50-100 "micro-CNVs" (ie 10-100 kb copy number variants, fig S4), and 290 indels: >"We call an SNV if there is a called NR allele and: (a) the total read depth in the bulk sample is above 15; (b) no bulk read has the NR allele; (c) if two SNVs are within 100bp from each other, both are discarded. With this procedure, we called 15,940 SNVs on the autosome of sample BJ1...We called 294 putative INDELs from the BJ1-BJ2 pair, the negative control" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538131/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538131/ So taking the smallest and biggest numbers of possible here gives: 100%*(15e3 + 50*10e3 + 290*1)/3e9 = 0.02% of bases affected 100%*(15e3 + 100*100e3 + 290*10e3)/3e9 = 0.4% of bases affected This seems to ignore any very small or large mutations that may affect a given site, and not all the mutations will simulate the effect of NHEJ... but this makes me think it isnt impossible for 0.001-1% to happen, especially in vitro.
  - nonbel 8y ago
    Ok, I guess no one calls it an indel if theres no net change to the length. It doesnt really matter to the example if its an indel or substitution.

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.
    - nonbel 8y ago
      Here is what I am responding to (also keep in mind I was using indel to mean "indels and substitutions", ie whatever may be counted as an error during NHEJ): > "There's no way cells survive with indels at 10% of their base positions, that's not possible." > "How is the per-base indel rate not 10% if 10% of cells carry an indel at any given locus?" I figured you were referring to per base within each cell. Your overall "per base" rate is getting the average (across cells) number of mutated sites. The survival of a cell is determined by its own genome, not the average of the population its been grouped into. However, it is the case that if x% of cells have a mutation at any given site then there must be at least some cells in this group that contain x% or greater mutated sites. Is that what you meant? Because that's not a bad point. I don't know where that 10% value came from and have no attachment to it, just assume that one is measurement noise for now, but even cells with 0.1% of sites mutated seems like a lot. There doesn't seem to be many whole genome single cell sequencing results available yet, which is what I think is needed here. Here is one that reports ~ 15k SNVs, 50-100 "micro-CNVs" (ie 10-100 kb copy number variants, fig S4), and 290 indels: >"We call an SNV if there is a called NR allele and: (a) the total read depth in the bulk sample is above 15; (b) no bulk read has the NR allele; (c) if two SNVs are within 100bp from each other, both are discarded. With this procedure, we called 15,940 SNVs on the autosome of sample BJ1...We called 294 putative INDELs from the BJ1-BJ2 pair, the negative control" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538131/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538131/ So taking the smallest and biggest numbers of possible here gives: 100%*(15e3 + 50*10e3 + 290*1)/3e9 = 0.02% of bases affected 100%*(15e3 + 100*100e3 + 290*10e3)/3e9 = 0.4% of bases affected This seems to ignore any very small or large mutations that may affect a given site, and not all the mutations will simulate the effect of NHEJ... but this makes me think it isnt impossible for 0.001-1% to happen, especially in vitro.
- nonbel 8y ago
  Ok, I guess no one calls it an indel if theres no net change to the length. It doesnt really matter to the example if its an indel or substitution.