Author: David Gerard

# Simulate Next-Generation Sequencing Data

#### David Gerard

# Abstract

We demonstrate how to simulate NGS data under various genotype distributions, then fit these data using `flexdog`. The genotyping methods are described in Gerard et al. (2018).

# Analysis

Let’s suppose that we have 100 hexaploid individuals, with varying levels of read-depth.

    set.seed(1)
    library(updog)
    nind    <- 100
    ploidy  <- 6
    sizevec <- round(stats::runif(n   = nind,
                                  min = 50,
                                  max = 200))

We can simulate their read-counts under various genotype distributions, allele biases, overdispersions, and sequencing error rates using the `rgeno` and `rflexdog` functions.

## F1 Population

Suppose these individuals are all siblings where the first parent has 4 copies of the reference allele and the second parent has 5 copies of the reference allele. Then the following code, using `rgeno`, will simulate the individuals’ genotypes.

    true_geno <- rgeno(n      = nind,
                       ploidy = ploidy,
                       model  = "f1",
                       p1geno = 4,
                       p2geno = 5)

Once we have their genotypes, we can simulate their read-counts using `rflexdog`. Let’s suppose that there is a moderate level of allelic bias (0.7) and a small level of overdispersion (0.005). Generally, in the real data that I’ve seen, the bias will range between 0.5 and 2 and the overdispersion will range between 0 and 0.02, with only a few extremely overdispersed SNPs above 0.02.

    refvec <- rflexdog(sizevec = sizevec,
                       geno    = true_geno,
                       ploidy  = ploidy,
                       seq     = 0.001,
                       bias    = 0.7,
                       od      = 0.005)

When we plot the data, it looks realistic

    plot_geno(refvec  = refvec,
              sizevec = sizevec,
              ploidy  = ploidy,
              bias    = 0.7,
              seq     = 0.001,
              geno    = true_geno)

We can test `flexdog` on these data

    fout <- flexdog(refvec  = refvec,
                    sizevec = sizevec,
                    ploidy  = ploidy,
                    model   = "f1")
    #>          Fit: 1 of 5
    #> Initial Bias: 0.3678794
    #> Log-Likelihood: -364.3851
    #> Keeping new fit.
    #>
    #>          Fit: 2 of 5
    #> Initial Bias: 0.6065307
    #> Log-Likelihood: -364.3851
    #> Keeping old fit.
    #>
    #>          Fit: 3 of 5
    #> Initial Bias: 1
    #> Log-Likelihood: -364.385
    #> Keeping new fit.
    #>
    #>          Fit: 4 of 5
    #> Initial Bias: 1.648721
    #> Log-Likelihood: -381.6985
    #> Keeping old fit.
    #>
    #>          Fit: 5 of 5
    #> Initial Bias: 2.718282
    #> Log-Likelihood: -412.5899
    #> Keeping old fit.
    #>
    #> Done!

`flexdog` gives us reasonable genotyping, and it accurately estimates the proportion of individuals mis-genotyped.

    plot(fout)

    ## Estimated proportion misgenotyped
    fout$prop_mis
    #> [1] 0.06990052

    ## Actual proportion misgenotyped
    mean(fout$geno != true_geno)
    #> [1] 0.05

## HWE Population

Now run the same simulations assuming the individuals are in Hardy-Weinberg population with an allele frequency of 0.75.

    true_geno <- rgeno(n      = nind,
                       ploidy = ploidy,
                       model  = "hw",
                       allele_freq = 0.75)
    refvec <- rflexdog(sizevec = sizevec,
                       geno    = true_geno,
                       ploidy  = ploidy,
                       seq     = 0.001,
                       bias    = 0.7,
                       od      = 0.005)
    fout <- flexdog(refvec  = refvec,
                    sizevec = sizevec,
                    ploidy  = ploidy,
                    model   = "hw")
    #>          Fit: 1 of 5
    #> Initial Bias: 0.3678794
    #> Log-Likelihood: -378.2634
    #> Keeping new fit.
    #>
    #>          Fit: 2 of 5
    #> Initial Bias: 0.6065307
    #> Log-Likelihood: -378.2633
    #> Keeping new fit.
    #>
    #>          Fit: 3 of 5
    #> Initial Bias: 1
    #> Log-Likelihood: -378.2633
    #> Keeping old fit.
    #>
    #>          Fit: 4 of 5
    #> Initial Bias: 1.648721
    #> Log-Likelihood: -378.2633
    #> Keeping old fit.
    #>
    #>          Fit: 5 of 5
    #> Initial Bias: 2.718282
    #> Log-Likelihood: -378.2634
    #> Keeping old fit.
    #>
    #> Done!
    plot(fout)

    ## Estimated proportion misgenotyped
    fout$prop_mis
    #> [1] 0.07603339

    ## Actual proportion misgenotyped
    mean(fout$geno != true_geno)
    #> [1] 0.07

    ## Estimated allele frequency close to true allele frequency
    fout$par$alpha
    #> [1] 0.7474485

# References

Gerard, David, Luís Felipe Ventorim Ferrão, Antonio Augusto Franco Garcia, and Matthew Stephens. 2018. “Genotyping Polyploids from Messy Sequencing Data.” Genetics 210 (3). Genetics: 789–807. https://doi.org/10.1534/genetics.118.301468.