Molecular Inversion Probes for targeted resequencing in non-model organisms

Applications that require resequencing of hundreds or thousands of predefined genomic regions in numerous samples are common in studies of non-model organisms. However few approaches at the scale intermediate between multiplex PCR and sequence capture methods are available. Here we explored the utility of Molecular Inversion Probes (MIPs) for the medium-scale targeted resequencing in a non-model system. Markers targeting 112 bp of exonic sequence were designed from transcriptome of Lissotriton newts. We assessed performance of 248 MIP markers in a sample of 85 individuals. Among the 234 (94.4%) successfully amplified markers 80% had median coverage within one order of magnitude, indicating relatively uniform performance; coverage uniformity across individuals was also high. In the analysis of polymorphism and segregation within family, 77% of 248 tested MIPs were confirmed as single copy Mendelian markers. Genotyping concordance assessed using replicate samples exceeded 99%. MIP markers for targeted resequencing have a number of advantages: high specificity, high multiplexing level, low sample requirement, straightforward laboratory protocol, no need for preparation of genomic libraries and no ascertainment bias. We conclude that MIP markers provide an effective solution for resequencing targets of tens or hundreds of kb in any organism and in a large number of samples

Development of a single nucleotide polymorphism array for population genomic studies in four European pine species

Pines are some of the most ecologically and economically important tree species in the world, and many have enormous natural distributions or have been extensively planted. However, a lack of rapid genotyping capability is hampering progress in understanding the molecular basis of genetic variation in these species. Here, we deliver an efficient tool for genotyping thousands of single nucleotide polymorphism (SNP) markers across the genome that can be applied to genetic studies in pines. Polymorphisms from resequenced candidate genes and transcriptome sequences of P. sylvestris, P. mugo, P. uncinata, P. uliginosa and P. radiata were used to design a 49,829 SNP array (Axiom_PineGAP, Thermo Fisher). Over a third (34.68%) of the unigenes identified from the P. sylvestris transcriptome were represented on the array, which was used to screen samples of four pine species. The conversion rate for the array on all samples was 42% (N = 20,795 SNPs) and was similar for SNPs sourced from resequenced candidate gene and transcriptome sequences. The broad representation of gene ontology terms by unigenes containing converted SNPs reflected their coverage across the full transcriptome. Over a quarter of successfully converted SNPs were polymorphic among all species, and the data were successful in discriminating among the species and some individual populations. The SNP array provides a valuable new tool to advance genetic studies in these species and demonstrates the effectiveness of the technology for rapid genotyping in species with large and complex genomes

A draft fur seal genome provides insights into factors affecting SNP validation and how to mitigate them

Custom genotyping arrays provide a flexible and accurate means of genotyping single nucleotide polymorphisms (SNPs) in a large number of individuals of essentially any organism. However, validation rates, defined as the proportion of putative SNPs that are verified to be polymorphic in a population, are often very low. A number of potential causes of assay failure have been identified, but none have been explored systematically. In particular, as SNPs are often developed from transcriptomes, parameters relating to the genomic context are rarely taken into account. Here, we assembled a draft Antarctic fur seal (Arctocephalus gazella) genome (assembly size: 2.41Gb; scaffold/contig N50: 3.1Mb/27.5kb). We then used this resource to map the probe sequences of 144 putative SNPs genotyped in 480 individuals. The number of probe-to-genome mappings and alignment length together explained almost a third of the variation in validation success, indicating that sequence uniqueness and proximity to intron-exon boundaries play an important role. The same pattern was found after mapping the probe sequences to the Walrus and Weddell seal genomes, suggesting that the genomes of species divergent by as much as 23 million years can hold information relevant to SNP validation outcomes. Additionally, re-analysis of genotyping data from seven previous studies found the same two variables to be significantly associated with SNP validation success across a variety of taxa. Finally, our study reveals considerable scope for validation rates to be improved, either by simply filtering for SNPs whose flanking sequences align uniquely and completely to a reference genome, or through predictive modeling

Population Genetics And Mixed Stock Analysis Of Chum Salmon (Oncorhynchus Keta) With Molecular Genetics

Thesis (Ph.D.) University of Alaska Fairbanks, 2012Chum salmon (Oncorhynchus keta) are important for subsistence and commercial harvest in Alaska. Variability of returns to western Alaskan drainages that caused economic hardship for stakeholders has led to speculation about reasons, which may include both anthropogenic and environmental causes in the marine environment. Mixed stock analysis (MSA) compares genetic information from an individual caught at sea to a reference baseline of genotypes to assign it to its population of origin. Application of genetic baselines requires several complex steps that can introduce bias. The bias may reduce the accuracy of MSA and result in overly-optimistic evaluations of baselines. Moreover, some applications that minimize bias cannot use informative haploid mitochondrial variation. Costs of baseline development are species-specific and difficult to predict. Finally, because populations of western Alaskan chum salmon demonstrate weak genetic divergence, samples from mixtures cannot be accurately assigned to a population of origin. The chapters of this thesis address three challenges. The first chapter describes technical aspects of genetic marker development. The second chapter describes a method to evaluate the precision and accuracy of a genetic baseline that accepts any type of data and reduces bias that may have been introduced during baseline development. This chapter also includes a method that places a cost on baseline development by predicting the number of markers needed to achieve a given accuracy. The final chapter explores the reasons for the weak genetic structure of western Alaskan chum salmon populations. The results of those analyses and both geological and archaeological data suggest that recent environmental and geological processes may be involved. The methods and analyses in this thesis can be applied to any species and may be particularly useful for other western Alaskan species