High throughput crop genome genotyping by a combination of pool next generation sequencing and haplotype-based data processing

BackgroundThe identification of environmentally specific alleles and the observation of evolutional processes is a goal of conservation genomics. By generational changes of allele frequencies in populations, questions regarding effective population size, gene flow, drift, and selection can be addressed. The observation of such effects often is a trade-off of costs and resolution, when a decent sample of genotypes should be genotyped for many loci. Pool genotyping approaches can derive a high resolution and precision in allele frequency estimation, when high coverage sequencing is utilized. Still, pool high coverage pool sequencing of big genomes comes along with high costs.ResultsHere we present a reliable method to estimate a barley population’s allele frequency at low coverage sequencing. Three hundred genotypes were sampled from a barley backcross population to estimate the entire population’s allele frequency. The allele frequency estimation accuracy and yield were compared for three next generation sequencing methods. To reveal accurate allele frequency estimates on a low coverage sequencing level, a haplotyping approach was performed. Low coverage allele frequency of positional connected single polymorphisms were aggregated to a single haplotype allele frequency, resulting in two to 271 times higher depth and increased precision. We compared different haplotyping tactics, showing that gene and chip marker-based haplotypes perform on par or better than simple contig haplotype windows. The comparison of multiple pool samples and the referencing against an individual sequencing approach revealed whole genome pool resequencing having the highest correlation to individual genotyping (up to 0.97), while transcriptomics and genotyping by sequencing indicated higher error rates and lower correlations.ConclusionUsing the proposed method allows to identify the allele frequency of populations with high accuracy at low cost. This is particularly interesting for conservation genomics in species with big genomes, like barley or wheat. Whole genome low coverage resequencing at 10x coverage can deliver a highly accurate estimation of the allele frequency, when a loci-based haplotyping approach is applied. Using annotated haplotypes allows to capitalize from biological background and statistical robustness.