Estimating the Human Mutation Rate: Direct Method

This is the fourth in a series of posts on human mutation rates and their implication(s). The first three were ...

What Is a Mutation?
Estimating the Human Mutation Rate: Biochemical Method
Estimating the Human Mutation Rate: Phylogenetic Method

There are basically three ways to estimate the mutation rate in the human lineage. I refer to them as the Biochemical Method, the Phylogenetic Method, and the Direct Method.

The Biochemical Method is based on our knowledge of biochemistry and DNA replication as well as estimates of the number of cell divisions between zygote and egg. It gives a value of 130 mutations per generation. The Phylogenetic Method depends on the fact that most mutations are neutral and that the rate of fixation of alleles is equal to the mutation rate. It also relies on a correct phylogeny. The Phylogenetic Method gives values between 112-160 mutations per generation. These two methods are pretty much in agreement.

The Direct Method involves sequencing the entire genomes of related individuals (e.g. mother, father, child) and simply counting the new mutations in the offspring. You might think that the Direct Method gives a definitive result that doesn't rely on any assumptions, therefore it should yield the most accurate result. The other two methods should be irrelevant.

This would be true if the Direct Method were as easy as it sounds but things are more complicated.

The first paper to be published was by Xue et al. (2009). They looked at the sequences of Y chromosomes from two men separated by 13 generations. (6 generations in one lineage and 7 generations in the other.) The Y chromosomes differed by four mutations in 10.15 × 10⁶ bp.¹ These are neutral mutations and the rate works out to 3.0 × 10^-8 mutations per base pair per generation.

If we assume an average of 400 cell divisions per generation (male lineage) then this gives a mutation rate of 0.75 × 10^-10 mutations per bp per replication. This isn't far from the value of 1.0 × 10^-10 that we used in the Biochemical Method.

If we apply this mutation rate to the entire genome then there will be 96 mutations in each sperm cell and 7 in each egg cell for a total of ...

103 mutations per generation

Theme

Mutation

-definition
-mutation types
-mutation rates
-phylogeny
-controversies

The problems with this calculation have to do deciding how many real mutations there are. In this particular experiment, the Y chromosomes were extracted from cells in culture. The authors actually found 23 differences between the two Y chromosomes but only 12 of these were confirmed by resequencing. Of these, only four were confirmed by sequencing DNA directly from the donors. (Eight mutations occurred during growth of the cell lines.) The authors are confident that they have not missed any mutations and I suspect that the number of false negatives is, in fact, close to zero.

This value (103 mutations per generation) is on the low end of the values calculated previously but the error bars are significant due to the low number of mutations.

Three other papers have appeared recently.²

1. Roach et al. (2012) sequenced genomes from a family of four (mother, father, two children). They found 33,937 potential mutations but confirmed only 28 mutations in the two children. After making some adjustments for false negatives they estimate that the total average number of mutations per diploid genome per generation was ...

70 mutations per generation

This is about half the value estimated by the Biochemical and Phylogenetic Methods. It's not clear to me how they estimated the true number of mutations. What is clear is that it is not easy to count mutations when dealing with sloppy sequences.

2. Conrad et al. (2011) looked at two sets of parents and offspring (trios). They used cell lines so they had to distinguish between germline mutations and somatic cell mutations. One of the offspring had 49 mutations and the other had 35 mutations. There were 1,586 somatic cell mutations that had to be eliminated. After correcting for false negatives, they estimate 60 mutations in one child and 45 mutations in the other. Since only 2.555 Gb were analyzed, this works out to ...

75 mutations per generation
56 mutations per generation

These values are lower than what we expected from previous studies. The authors determined that 92% of the mutations in one offspring were from the father but only 36% of the mutations in the other trio were from the father. This is not reasonable and neither is the discrepancy in total mutations between the two different offspring. It suggests that there are a lot of errors in this study.

3. The most comprehensive study so far is from Kong et al. (2012). These authors looked at 78 Icelandic families whose genealogies were well known. They sequenced the genomes of 219 distinct individuals and found an average of 63.2 mutations in each child. Since they only looked at 2.63 Gb, this translates to ...

77 mutations per generation

Individual values vary over a wide range. The lowest score reported is 58 and the highest is 129. This study suffers from the same problems as the other two direct sequencing experiments; namely, that it's difficult to decide which of the differences are real mutations and which ones are artifacts. The authors claim that their false negative rate is only 2%.

The whole genome sequencing papers have been widely reported as giving a result that is half the mutation rate we estimated previously. This is a problem because the mutation rate is used in many calculations. We'll discuss the implications in later posts.

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1. The Y chromosome is 24 Mb but they couldn't analyze regions of repeats and some other regions weren't well covered.

2. Please let me know if I missed any papers.

Conrad, D.F., et al. (2011) Variation in genome-wide mutation rates within and between human families. Nature Genetics 43:712-715. [doi: 10.1038/ng.862]

Kong, A., et al. (2012) Rate of de novo mutations and the importance of father's age to disease risk. Nature 488:471-475. [doi: 10.1038/nature11396]

Roach, J.C., et al. (2010) Analysis of genetic inheritance in a family quartet by whole-genome sequencing. Science 328:636-639. [doi: 10.1126/science.1186802]

Xue, Y., et al. (2009) Human Y chromosome base-substitution mutation rate measured by direct sequencing in a deep-rooting pedigree. Current Biology 19:1453-1457. [doi: 10.1016/j.cub.2009.07.032]