William A. Cresko

Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F2 individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms.

Population Genomics of Parallel Adaptation in Threespine Stickleback using Sequenced RAD Tags

Next-generation sequencing technology provides novel opportunities for gathering genome-scale sequence data in natural populations, laying the empirical foundation for the evolving field of population genomics. Here we conducted a genome scan of nucleotide diversity and differentiation in natural populations of threespine stickleback (Gasterosteus aculeatus). We used Illumina-sequenced RAD tags to identify and type over 45,000 single nucleotide polymorphisms (SNPs) in each of 100 individuals from two oceanic and three freshwater populations. Overall estimates of genetic diversity and differentiation among populations confirm the biogeographic hypothesis that large panmictic oceanic populations have repeatedly given rise to phenotypically divergent freshwater populations. Genomic regions exhibiting signatures of both balancing and divergent selection were remarkably consistent across multiple, independently derived populations, indicating that replicate parallel phenotypic evolution in stickleback may be occurring through extensive, parallel genetic evolution at a genome-wide scale. Some of these genomic regions co-localize with previously identified QTL for stickleback phenotypic variation identified using laboratory mapping crosses. In addition, we have identified several novel regions showing parallel differentiation across independent populations. Annotation of these regions revealed numerous genes that are candidates for stickleback phenotypic evolution and will form the basis of future genetic analyses in this and other organisms. This study represents the first high-density SNP–based genome scan of genetic diversity and differentiation for populations of threespine stickleback in the wild. These data illustrate the complementary nature of laboratory crosses and population genomic scans by confirming the adaptive significance of previously identified genomic regions, elucidating the particular evolutionary and demographic history of such regions in natural populations, and identifying new genomic regions and candidate genes of evolutionary significance.

Stacks: an analysis tool set for population genomics

Massively parallel short‐read sequencing technologies, coupled with powerful software platforms, are enabling investigators to analyse tens of thousands of genetic markers. This wealth of data is rapidly expanding and allowing biological questions to be addressed with unprecedented scope and precision. The sizes of the data sets are now posing significant data processing and analysis challenges. Here we describe an extension of the Stacks software package to efficiently use genotype‐by‐sequencing data for studies of populations of organisms. Stacks now produces core population genomic summary statistics and SNP‐by‐SNP statistical tests. These statistics can be analysed across a reference genome using a smoothed sliding window. Stacks also now provides several output formats for several commonly used downstream analysis packages. The expanded population genomics functions in Stacks will make it a useful tool to harness the newest generation of massively parallel genotyping data for ecological and evolutionary genetics.

Stacks: Building and Genotyping Loci De Novo From Short-Read Sequences

Advances in sequencing technology provide special opportunities for genotyping individuals with speed and thrift, but the lack of software to automate the calling of tens of thousands of genotypes over hundreds of individuals has hindered progress. Stacks is a software system that uses short-read sequence data to identify and genotype loci in a set of individuals either de novo or by comparison to a reference genome. From reduced representation Illumina sequence data, such as RAD-tags, Stacks can recover thousands of single nucleotide polymorphism (SNP) markers useful for the genetic analysis of crosses or populations. Stacks can generate markers for ultra-dense genetic linkage maps, facilitate the examination of population phylogeography, and help in reference genome assembly. We report here the algorithms implemented in Stacks and demonstrate their efficacy by constructing loci from simulated RAD-tags taken from the stickleback reference genome and by recapitulating and improving a genetic map of the zebrafish, Danio rerio.

Resolving postglacial phylogeography using high-throughput sequencing

The distinction between model and nonmodel organisms is becoming increasingly blurred. High-throughput, second-generation sequencing approaches are being applied to organisms based on their interesting ecological, physiological, developmental, or evolutionary properties and not on the depth of genetic information available for them. Here, we illustrate this point using a low-cost, efficient technique to determine the fine-scale phylogenetic relationships among recently diverged populations in a species. This application of restriction site-associated DNA tags (RAD tags) reveals previously unresolved genetic structure and direction of evolution in the pitcher plant mosquito, Wyeomyia smithii, from a southern Appalachian Mountain refugium following recession of the Laurentide Ice Sheet at 22,000–19,000 B.P. The RAD tag method can be used to identify detailed patterns of phylogeography in any organism regardless of existing genomic data, and, more broadly, to identify incipient speciation and genome-wide variation in natural populations in general.

SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing

Next-generation sequencing technologies are revolutionizing the field of evolutionary biology, opening the possibility for genetic analysis at scales not previously possible. Research in population genetics, quantitative trait mapping, comparative genomics, and phylogeography that was unthinkable even a few years ago is now possible. More importantly, these next-generation sequencing studies can be performed in organisms for which few genomic resources presently exist. To speed this revolution in evolutionary genetics, we have developed Restriction site Associated DNA (RAD) genotyping, a method that uses Illumina next-generation sequencing to simultaneously discover and score tens to hundreds of thousands of single-nucleotide polymorphism (SNP) markers in hundreds of individuals for minimal investment of resources. In this chapter, we describe the core RAD-seq protocol, which can be modified to suit a diversity of evolutionary genetic questions. In addition, we discuss bioinformatic considerations that arise from unique aspects of next-generation sequencing data as compared to traditional marker-based approaches, and we outline some general analytical approaches for RAD-seq and similar data. Despite considerable progress, the development of analytical tools remains in its infancy, and further work is needed to fully quantify sampling variance and biases in these data types.

Extensive linkage disequilibrium and parallel adaptive divergence across threespine stickleback genomes

Population genomic studies are beginning to provide a more comprehensive view of dynamic genome-scale processes in evolution. Patterns of genomic architecture, such as genomic islands of increased divergence, may be important for adaptive population differentiation and speciation. We used next-generation sequencing data to examine the patterns of local and long-distance linkage disequilibrium (LD) across oceanic and freshwater populations of threespine stickleback, a useful model for studies of evolution and speciation. We looked for associations between LD and signatures of divergent selection, and assessed the role of recombination rate variation in generating LD patterns. As predicted under the traditional biogeographic model of unidirectional gene flow from ancestral oceanic to derived freshwater stickleback populations, we found extensive local and long-distance LD in fresh water. Surprisingly, oceanic populations showed similar patterns of elevated LD, notably between large genomic regions previously implicated in adaptation to fresh water. These results support an alternative biogeographic model for the stickleback radiation, one of a metapopulation with appreciable bi-directional gene flow combined with strong divergent selection between oceanic and freshwater populations. As predicted by theory, these processes can maintain LD within and among genomic islands of divergence. These findings suggest that the genomic architecture in oceanic stickleback populations may provide a mechanism for the rapid re-assembly and evolution of multi-locus genotypes in newly colonized freshwater habitats, and may help explain genetic mapping of parallel phenotypic variation to similar loci across independent freshwater populations.

Local De Novo Assembly of RAD Paired-End Contigs Using Short Sequencing Reads

Despite the power of massively parallel sequencing platforms, a drawback is the short length of the sequence reads produced. We demonstrate that short reads can be locally assembled into longer contigs using paired-end sequencing of restriction-site associated DNA (RAD-PE) fragments. We use this RAD-PE contig approach to identify single nucleotide polymorphisms (SNPs) and determine haplotype structure in threespine stickleback and to sequence E. coli and stickleback genomic DNA with overlapping contigs of several hundred nucleotides. We also demonstrate that adding a circularization step allows the local assembly of contigs up to 5 kilobases (kb) in length. The ease of assembly and accuracy of the individual contigs produced from each RAD site sequence suggests RAD-PE sequencing is a useful way to convert genome-wide short reads into individually-assembled sequences hundreds or thousands of nucleotides long.

Genome duplication, subfunction partitioning, and lineage divergence: Sox9 in stickleback and zebrafish

Teleosts are the most species‐rich group of vertebrates, and a genome duplication (tetraploidization) event in ray‐fin fish appears to have preceded this remarkable explosion of biodiversity. What is the relationship of the ray‐fin genome duplication to the teleost radiation? Genome duplication may have facilitated lineage divergence by partitioning different ancestral gene subfunctions among co‐orthologs of tetrapod genes in different teleost lineages. To test this hypothesis, we investigated gene expression patterns for Sox9 gene duplicates in stickleback and zebrafish, teleosts whose lineages diverged early in Euteleost evolution. Most expression domains appear to have been partitioned between Sox9a and Sox9b before the divergence of stickleback and zebrafish lineages, but some ancestral expression domains were distributed differentially in each lineage. We conclude that some gene subfunctions, as represented by lineage‐specific expression domains, may have assorted differently in separate lineages and that these may have contributed to lineage diversification during teleost evolution. Developmental Dynamics, 2003.

USING POPULATION GENOMICS TO DETECT SELECTION IN NATURAL POPULATIONS: KEY CONCEPTS AND METHODOLOGICAL CONSIDERATIONS

Natural selection shapes patterns of genetic variation among individuals, populations, and species, and it does so differentially across genomes. The field of population genomics provides a comprehensive genome-scale view of the action of selection, even beyond traditional model organisms. However, even with nearly complete genomic sequence information, our ability to detect the signature of selection on specific genomic regions depends on choosing experimental and analytical tools appropriate to the biological situation. For example, processes that occur at different timescales, such as sorting of standing genetic variation, mutation-selection balance, or fixed interspecific divergence, have different consequences for genomic patterns of variation. Inappropriate experimental or analytical approaches may fail to detect even strong selection or falsely identify a signature of selection. Here we outline the conceptual framework of population genomics, relate genomic patterns of variation to evolutionary processes, and identify major biological factors to be considered in studies of selection. As data-gathering technology continues to advance, our ability to understand selection in natural populations will be limited more by conceptual and analytical weaknesses than by the amount of molecular data. Our aim is to bring critical biological considerations to the fore in population genomics research and to spur the development and application of analytical tools appropriate to diverse biological systems.