Population structure of Atlantic mackerel inferred from RAD‐seq‐derived SNP markers: effects of sequence clustering parameters and hierarchical SNP selection

Restriction‐site‐associated DNA sequencing (RAD‐seq) and related methods are revolutionizing the field of population genomics in nonmodel organisms as they allow generating an unprecedented number of single nucleotide polymorphisms (SNPs) even when no genomic information is available. Yet, RAD‐seq data analyses rely on assumptions on nature and number of nucleotide variants present in a single locus, the choice of which may lead to an under‐ or overestimated number of SNPs and/or to incorrectly called genotypes. Using the Atlantic mackerel (Scomber scombrus L.) and a close relative, the Atlantic chub mackerel (Scomber colias), as case study, here we explore the sensitivity of population structure inferences to two crucial aspects in RAD‐seq data analysis: the maximum number of mismatches allowed to merge reads into a locus and the relatedness of the individuals used for genotype calling and SNP selection. Our study resolves the population structure of the Atlantic mackerel, but, most importantly, provides insights into the effects of alternative RAD‐seq data analysis strategies on population structure inferences that are directly applicable to other species.     

Cross‐platform compatibility of de novo‐aligned SNPs in a nonmodel butterfly genus

High‐throughput sequencing methods for genotyping genome‐wide markers are being rapidly adopted for phylogenetics of nonmodel organisms in conservation and biodiversity studies. However, the reproducibility of SNP genotyping and degree of marker overlap or compatibility between datasets from different methodologies have not been tested in nonmodel systems. Using double‐digest restriction site‐associated DNA sequencing, we sequenced a common set of 22 specimens from the butterfly genus Speyeria on two different Illumina platforms, using two variations of library preparation. We then used a de novo approach to bioinformatic locus assembly and SNP discovery for subsequent phylogenetic analyses. We found a high rate of locus recovery despite differences in library preparation and sequencing platforms, as well as overall high levels of data compatibility after data processing and filtering. These results provide the first application of NGS methods for phylogenetic reconstruction in Speyeria and support the use and long‐term viability of SNP genotyping applications in nonmodel systems.     

Small‐ and large‐scale heterogeneity in genetic variation across the collard flycatcher genome: implications for estimating genetic diversity in nonmodel organisms

Population genetic studies in nonmodel organisms are often hampered by a lack of reference genomes that are essential for whole‐genome resequencing. In the light of this, genotyping methods have been developed to effectively eliminate the need for a reference genome, such as genotyping by sequencing or restriction site‐associated DNA sequencing (RAD‐seq). However, what remains relatively poorly studied is how accurately these methods capture both average and variation in genetic diversity across an organism's genome. In this issue of Molecular Ecology Resources, Dutoit et al. (2016) use whole‐genome resequencing data from the collard flycatcher to assess what factors drive heterogeneity in nucleotide diversity across the genome. Using these data, they then simulate how well different sequencing designs, including RAD sequencing, could capture most of the variation in genetic diversity. They conclude that for evolutionary and conservation‐related studies focused on the estimating genomic diversity, researchers should emphasize the number of loci analysed over the number of individuals sequenced.     

Substantial differences in bias between single‐digest and double‐digest RAD‐seq libraries: A case study

The trade‐offs of using single‐digest vs. double‐digest restriction site‐associated DNA sequencing (RAD‐seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD‐seq. Sixty individuals were subjected to single‐digest RAD‐seq (sdRAD‐seq), and the remaining 384 individuals were genotyped using a double‐digest RAD‐seq (ddRAD‐seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD‐seq than with ddRAD‐seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD‐seq versus ddRAD‐seq. Our finding that sdRAD‐seq and ddRAD‐seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations.     

High‐throughput sequencing and graph‐based cluster analysis facilitate microsatellite development from a highly complex genome

Despite recent advances in high‐throughput sequencing, difficulties are often encountered when developing microsatellites for species with large and complex genomes. This probably reflects the close association in many species of microsatellites with cryptic repetitive elements. We therefore developed a novel approach for isolating polymorphic microsatellites from the club‐legged grasshopper (Gomphocerus sibiricus), an emerging quantitative genetic and behavioral model system. Whole genome shotgun Illumina MiSeq sequencing was used to generate over three million 300 bp paired‐end reads, of which 67.75% were grouped into 40,548 clusters within RepeatExplorer. Annotations of the top 468 clusters, which represent 60.5% of the reads, revealed homology to satellite DNA and a variety of transposable elements. Evaluating 96 primer pairs in eight wild‐caught individuals, we found that primers mined from singleton reads were six times more likely to amplify a single polymorphic microsatellite locus than primers mined from clusters. Our study provides experimental evidence in support of the notion that microsatellites associated with repetitive elements are less likely to successfully amplify. It also reveals how advances in high‐throughput sequencing and graph‐based repetitive DNA analysis can be leveraged to isolate polymorphic microsatellites from complex genomes.     

Next‐generation sequencing sheds light on intricate regulation of insect gut microbiota

Next‐generation sequencing (NGS) technologies are getting cheaper and easier and hence becoming readily accessible for many researchers in biological disciplines including ecology. In this issue of Molecular Ecology, Sudakaran et al. (2012) show how the NGS revolution contributes to our better and more comprehensive understanding of ecological interactions between gut symbiotic microbiota and the host organism. Using the European red firebug Pyrrhocoris apterus as a model system, they demonstrated that the gut microbiota consists of a small number of major bacterial phylotypes plus other minor bacterial associates. The major bacteria are localized in a specific anoxic section of the midgut and quantitatively account for most of the gut microbiota irrespective of host's geographic populations. The specific gut microbiota is established through early nymphal development of the host insect. Interestingly, the host feeding on different food, namely linden seeds, sunflower seeds or wasp larvae, scarcely affected the symbiont composition, suggesting homoeostatic control over the major symbiotic microbiota in the anoxic section of the midgut. Some of the minor components of the gut microbiota, which conventional PCR/cloning/sequencing approaches would have failed to detect, were convincingly shown to be food‐derived. These findings rest on the robust basis of high‐throughput sequencing data, and some of them could not be practically obtained by conventional molecular techniques, highlighting the significant impact of NGS approaches on ecological aspects of host–symbiont interactions in a nonmodel organism.     

Characterization of 20 microsatellite marker loci in Coquerel's sifaka (Propithecus coquereli)

The Coquerel's sifaka (Propithecus coquereli) is one of the species of Propithecus, living in the dry forest of northwest Madagascar. This species is endangered due to the loss and fragmentation of its natural habitat, consequential of deforestation. Twenty nuclear microsatellite loci were isolated from genomic DNA derived from a free ranging Coquerel's sifaka from the Mariarano Classified Forest, Madagascar. Population genetic parameters were estimated as baseline values from samples collected in that forest.