RADseq underestimates diversity and introduces genealogical biases due to nonrandom haplotype sampling

Abstract

Reduced representation genome-sequencing approaches based on restriction digestion are enabling large-scale marker generation and facilitating genomic studies in a wide range of model and nonmodel systems. However, sampling chromosomes based on restriction digestion may introduce a bias in allele frequency estimation due to polymorphisms in restriction sites. To explore the effects of this nonrandom sampling and its sensitivity to different evolutionary parameters, we developed a coalescent-simulation framework to mimic the biased recovery of chromosomes in restriction-based short-read sequencing experiments (RADseq). We analysed simulated DNA sequence datasets and compared known values from simulations with those that would be estimated using a RADseq approach from the same samples. We compare these 'true' and 'estimated' values of commonly used summary statistics, π, θ(w), Tajima's D and F(ST). We show that loci with missing haplotypes have estimated summary statistic values that can deviate dramatically from true values and are also enriched for particular genealogical histories. These biases are sensitive to nonequilibrium demography, such as bottlenecks and population expansion. In silico digests with 102 completely sequenced Drosophila melanogaster genomes yielded results similar to our findings from coalescent simulations. Though the potential of RADseq for marker discovery and trait mapping in nonmodel systems remains undisputed, our results urge caution when applying this technique to make population genetic inferences.

Exploring the phylogeography of a hexaploid freshwater fish by RAD sequencing

“…Some predicted drawbacks of this method are the inability of the process to combine stacks of reads as longer contigs, with consequent reduced downstream applications, and potential for bias in estimates of population parameters (Arnold et al., 2013; DaCosta & Sorenson, 2014). In practice, we did not observe these limitations, our mapping showed more extensive coverage than anticipated, and this was reflected in the number of stacks retrieved.…”

“…The difference in missing data observed between these datasets must be due to removing SNPs which contain missing data for entire populations in the latter dataset. This suggests that most missing data we observe are due to mutations within one of the restriction sites leading to locus dropout at the population level (Arnold et al., 2013). These missing data are therefore not due to technical errors or low coverage, but real biological signal from private population mutations.…”

“…This approach results in higher repeatability, better control over genome coverage, greater sharing of sequenced fragments, and similar sequence read proportions across individuals (Peterson et al., 2012). The additional restriction digestion may also introduce artifacts; however, as mutations in restriction sites may result in underestimation of diversity due to allele dropout (Arnold, Corbett‐Detig, Hartl, & Bomblies, 2013). Indels, combined with stringent size selection, may also result in loci being dropped or included in particular individuals or populations during ddRAD sequencing (DaCosta & Sorenson, 2014).…”

Double‐digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles

“…Furthermore, the mutation risk hypothesis suggests that fewer homologous or shared loci will be recovered for loci with greater risk of mutations. Hence, longer MiSeq ddRAD reads, which have greater likelihood of mutations that create novel restriction sites within reads, may have been less likely to be shared among species than within species (Arnold, Corbett‐Detig, Hartl, & Bomblies, 2013; Gautier et al., 2013). Simple mutations in restriction sites may also increase the read length variation for longer MiSeq reads compared to shorter HiSeq reads, but we saw some sequences with restriction sites incorporated in the middle of the sequence, suggesting that length variation can also be caused by inefficient enzyme digestions.…”

Genomic signatures of population bottleneck and recovery in Northwest Atlantic pinnipeds

“…While both datasets included similar numbers of loci and sequences per individual, many more polymorphic loci and SNPs were identified among gray seal loci than harbor seal loci (Figure 4). We acknowledge that there exist several sources of bias in estimates of genetic diversity from RAD sequencing data (Arnold, Corbett‐Detig, Hartl, & Bomblies, 2013; Cariou, Duret, & Charlat, 2016a,2016b; DaCosta & Sorenson, 2014; Davey et al., 2013; Gautier et al., 2013), but contend that relative comparisons of diversity between species for which data have been collected and processed in a similar manner should be robust (Lozier, 2014). …”