Diet assessment of the Atlantic Sea Nettle Chrysaora quinquecirrha in Barnegat Bay,New Jersey,using next‐generation sequencing

Next‐generation sequencing (NGS) methodologies have proven useful in deciphering the food items of generalist predators, but have yet to be applied to gelatinous animal gut and tentacle content. NGS can potentially supplement traditional methods of visual identification. Chrysaora quinquecirrha (Atlantic sea nettle) has progressively become more abundant in Mid‐Atlantic United States’ estuaries including Barnegat Bay (New Jersey), potentially having detrimental effects on both marine organisms and human enterprises. Full characterization of this predator's diet is essential for a comprehensive understanding of its impact on the food web and its management. Here, we tested the efficacy of NGS for prey item determination in the Atlantic sea nettle. We implemented a NGS ‘shotgun’ approach to randomly sequence DNA fragments isolated from gut lavages and gastric pouch/tentacle picks of eight and 84 sea nettles, respectively. These results were verified by visual identification and co‐occurring plankton tows. Over 550 000 contigs were assembled from ~110 million paired‐end reads. Of these, 100 contigs were confidently assigned to 23 different taxa, including soft‐bodied organisms previously undocumented as prey species, including copepods, fish, ctenophores, anemones, amphipods, barnacles, shrimp, polychaete worms, flukes, flatworms, echinoderms, gastropods, bivalves and hemichordates. Our results not only indicate that a ‘shotgun’ NGS approach can supplement visual identification methods, but targeted enrichment of a specific amplicon/gene is not a prerequisite for identifying Atlantic sea nettle prey items.     

exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

amplisas: a web server for multilocus genotyping using next‐generation amplicon sequencing data

Next‐generation sequencing (NGS) technologies are revolutionizing the fields of biology and medicine as powerful tools for amplicon sequencing (AS). Using combinations of primers and barcodes, it is possible to sequence targeted genomic regions with deep coverage for hundreds, even thousands, of individuals in a single experiment. This is extremely valuable for the genotyping of gene families in which locus‐specific primers are often difficult to design, such as the major histocompatibility complex (MHC). The utility of AS is, however, limited by the high intrinsic sequencing error rates of NGS technologies and other sources of error such as polymerase amplification or chimera formation. Correcting these errors requires extensive bioinformatic post‐processing of NGS data. Amplicon Sequence Assignment (amplisas ) is a tool that performs analysis of AS results in a simple and efficient way, while offering customization options for advanced users. amplisas is designed as a three‐step pipeline consisting of (i) read demultiplexing, (ii) unique sequence clustering and (iii) erroneous sequence filtering. Allele sequences and frequencies are retrieved in excel spreadsheet format, making them easy to interpret. amplisas performance has been successfully benchmarked against previously published genotyped MHC data sets obtained with various NGS technologies.     

DNA barcodes from century‐old type specimens using next‐generation sequencing

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next‐generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century‐old type specimens of Lepidoptera by attempting to recover 164‐bp and 94‐bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories – high (164‐bp sequence), medium (94‐bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR‐based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.     

Multiplex restriction amplicon sequencing: a novel next‐generation sequencing‐based marker platform for high‐throughput genotyping

To enable rapid selection of traits in marker‐assisted breeding, markers must be technically simple, low‐cost, high‐throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’‐ends, preceded by 6‐10 bases of specific or degenerate nucleotide sequences and then by a unique M13‐tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next‐generation sequencing‐based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

A targeted next‐generation sequencing toolkit for exon‐based cichlid phylogenomics

Cichlid fishes (family Cichlidae) are models for evolutionary and ecological research. Massively parallel sequencing approaches have been successfully applied to study relatively recent diversification in groups of African and Neotropical cichlids, but such technologies have yet to be used for addressing larger‐scale phylogenetic questions of cichlid evolution. Here, we describe a process for identifying putative single‐copy exons from five African cichlid genomes and sequence the targeted exons for a range of divergent (>tens of millions of years) taxa with probes designed from a single reference species (Oreochromis niloticus, Nile tilapia). Targeted sequencing of 923 exons across 10 cichlid species that represent the family's major lineages and geographic distribution resulted in a complete taxon matrix of 564 exons (649 549 bp), representing 559 genes. Maximum likelihood and Bayesian analyses in both species tree and concatenation frameworks yielded the same fully resolved and highly supported topology, which matched the expected backbone phylogeny of the major cichlid lineages. This work adds to the body of evidence that it is possible to use a relatively divergent reference genome for exon target design and successful capture across a broad phylogenetic range of species. Furthermore, our results show that the use of a third‐party laboratory coupled with accessible bioinformatics tools makes such phylogenomics projects feasible for research groups that lack direct access to genomic facilities. We expect that these resources will be used in further cichlid evolution studies and hope the protocols and identified targets will also be useful for phylogenetic studies of a wider range of organisms.     

High‐throughput SNP discovery in the rabbit (Oryctolagus cuniculus) genome by next‐generation semiconductor‐based sequencing

The European rabbit (Oryctolagus cuniculus) is a domesticated species with one of the broadest ranges of economic and scientific applications and fields of investigation. Rabbit genome information and assembly are available (oryCun2.0), but so far few studies have investigated its variability, and massive discovery of polymorphisms has not been published yet for this species. Here, we sequenced two reduced representation libraries (RRLs) to identify single nucleotide polymorphisms (SNPs) in the rabbit genome. Genomic DNA of 10 rabbits belonging to different breeds was pooled and digested with two restriction enzymes (HaeIII and RsaI) to create two RRLs which were sequenced using the Ion Torrent Personal Genome Machine. The two RRLs produced 2 917 879 and 4 046 871 reads, for a total of 280.51 Mb (248.49 Mb with quality >20) and 417.28 Mb (360.89 Mb with quality >20) respectively of sequenced DNA. About 90% and 91% respectively of the obtained reads were mapped on the rabbit genome, covering a total of 15.82% of the oryCun2.0 genome version. The mapping and ad hoc filtering procedures allowed to reliably call 62 491 SNPs. SNPs in a few genomic regions were validated by Sanger sequencing. The Variant Effect Predictor Web tool was used to map SNPs on the current version of the rabbit genome. The obtained results will be useful for many applied and basic research programs for this species and will contribute to the development of cost‐effective solutions for high‐throughput SNP genotyping in the rabbit.     

Genome‐wide sequence information reveals recurrent hybridization among diploid wheat wild relatives

Many conflicting hypotheses regarding the relationships among crops and wild species closely related to wheat (the genera Aegilops, Amblyopyrum, and Triticum) have been postulated. The contribution of hybridization to the evolution of these taxa is intensely discussed. To determine possible causes for this, and provide a phylogeny of the diploid taxa based on genome‐wide sequence information, independent data were obtained from genotyping‐by‐sequencing and a target‐enrichment experiment that returned 244 low‐copy nuclear loci. The data were analyzed using Bayesian, likelihood and coalescent‐based methods. D statistics were used to test if incomplete lineage sorting alone or together with hybridization is the source for incongruent gene trees. Here we present the phylogeny of all diploid species of the wheat wild relatives. We hypothesize that most of the wheat‐group species were shaped by a primordial homoploid hybrid speciation event involving the ancestral Triticum and Am. muticum lineages to form all other species except Ae. speltoides. This hybridization event was followed by multiple introgressions affecting all taxa except Triticum. Mostly progenitors of the extant species were involved in these processes, while recent interspecific gene flow seems insignificant. The composite nature of many genomes of wheat‐group taxa results in complicated patterns of diploid contributions when these lineages are involved in polyploid formation, which is, for example, the case for tetraploid and hexaploid wheats. Our analysis provides phylogenetic relationships and a testable hypothesis for the genome compositions in the basic evolutionary units within the wheat group of Triticeae.     

Haplotype‐based genotyping‐by‐sequencing in oat genome research

In a de novo genotyping‐by‐sequencing (GBS) analysis of short, 64‐base tag‐level haplotypes in 4657 accessions of cultivated oat, we discovered 164741 tag‐level (TL) genetic variants containing 241224 SNPs. From this, the marker density of an oat consensus map was increased by the addition of more than 70000 loci. The mapped TL genotypes of a 635‐line diversity panel were used to infer chromosome‐level (CL) haplotype maps. These maps revealed differences in the number and size of haplotype blocks, as well as differences in haplotype diversity between chromosomes and subsets of the diversity panel. We then explored potential benefits of SNP vs. TL vs. CL GBS variants for mapping, high‐resolution genome analysis and genomic selection in oats. A combined genome‐wide association study (GWAS) of heading date from multiple locations using both TL haplotypes and individual SNP markers identified 184 significant associations. A comparative GWAS using TL haplotypes, CL haplotype blocks and their combinations demonstrated the superiority of using TL haplotype markers. Using a principal component‐based genome‐wide scan, genomic regions containing signatures of selection were identified. These regions may contain genes that are responsible for the local adaptation of oats to Northern American conditions. Genomic selection for heading date using TL haplotypes or SNP markers gave comparable and promising prediction accuracies of up to r = 0.74. Genomic selection carried out in an independent calibration and test population for heading date gave promising prediction accuracies that ranged between r = 0.42 and 0.67. In conclusion, TL haplotype GBS‐derived markers facilitate genome analysis and genomic selection in oat.     

Unlocking the vault: next‐generation museum population genomics

Natural history museum collections provide unique resources for understanding how species respond to environmental change, including the abrupt, anthropogenic climate change of the past century. Ideally, researchers would conduct genome‐scale screening of museum specimens to explore the evolutionary consequences of environmental changes, but to date such analyses have been severely limited by the numerous challenges of working with the highly degraded DNA typical of historic samples. Here, we circumvent these challenges by using custom, multiplexed, exon capture to enrich and sequence ~11 000 exons (~4 Mb) from early 20th‐century museum skins. We used this approach to test for changes in genomic diversity accompanying a climate‐related range retraction in the alpine chipmunks (Tamias alpinus) in the high Sierra Nevada area of California, USA. We developed robust bioinformatic pipelines that rigorously detect and filter out base misincorporations in DNA derived from skins, most of which likely resulted from postmortem damage. Furthermore, to accommodate genotyping uncertainties associated with low‐medium coverage data, we applied a recently developed probabilistic method to call single‐nucleotide polymorphisms and estimate allele frequencies and the joint site frequency spectrum. Our results show increased genetic subdivision following range retraction, but no change in overall genetic diversity at either nonsynonymous or synonymous sites. This case study showcases the advantages of integrating emerging genomic and statistical tools in museum collection‐based population genomic applications. Such technical advances greatly enhance the value of museum collections, even where a pre‐existing reference is lacking and points to a broad range of potential applications in evolutionary and conservation biology.     

Discovering and sequencing new plant viral genomes by next‐generation sequencing: description of a practical pipeline

Small‐scale sequencing has improved substantially in recent decades, culminating in the development of next‐generation sequencing (NGS) technologies. Modern NGS methods have helped the discovery of many new plant viruses. Nevertheless, there is still a need to establish solid assembly pipelines targeting small genomes characterised by low identities to known viral sequences. Here, we describe and discuss the fundamental steps required for discovering and sequencing new plant viral genomes by NGS. A practical pipeline and standard alternative tools used in NGS analysis are presented.     

Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

High‐throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b‐RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b‐RAD protocols on non‐model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b‐RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade‐off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective‐base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b‐RAD protocols on non‐model organisms with different genome sizes, helping decision‐making for a reliable and cost‐effective genotyping.     

Efficient and sensitive identification and quantification of airborne pollen using next‐generation DNA sequencing

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen‐allergic patients. Current pollen monitoring methods are microscope‐based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost‐effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next‐generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.     

Development of genomic resources for Nothofagus species using next‐generation sequencing data

Using next‐generation sequencing, we developed the first whole‐genome resources for two hybridizing Nothofagus species of the Patagonian forests that crucially lack genomic data, despite their ecological and industrial value. A de novo assembly strategy combining base quality control and optimization of the putative chloroplast gene map yielded ~32 000 contigs from 43% of the reads produced. With 12.5% of assembled reads, we covered ~96% of the chloroplast genome and ~70% of the mitochondrial gene content, providing functional and structural annotations for 112 and 52 genes, respectively. Functional annotation was possible on 15% of the contigs, with ~1750 potentially novel nuclear genes identified for Nothofagus species. We estimated that the new resources (13.41 Mb in total) included ~4000 gene regions representing ~6.5% of the expected genic partition of the genome, the remaining contigs potentially being nongenic DNA. A high‐quality single nucleotide polymorphisms resource was developed by comparing various filtering methods, and preliminary results indicate a strong conservation of cpDNA genomes in contrast to numerous exclusive nuclear polymorphisms in both species. Finally, we characterized 2274 potential simple sequence repeat (SSR) loci, designed primers for 769 of them and validated nine of 29 loci in 42 individuals per species. Nothofagus obliqua had more alleles (4.89) on average than N. nervosa (2.89), 8 SSRs were efficient to discriminate species, and three were successfully transferred in three other Nothofagus species. These resources will greatly help for future inferences of demographic, adaptive and hybridizing events in Nothofagus species, and for conserving and managing natural populations.     

Genetic sex assignment in wild populations using genotyping‐by‐sequencing data: A statistical threshold approach

Establishing the sex of individuals in wild systems can be challenging and often requires genetic testing. Genotyping‐by‐sequencing (GBS) and other reduced‐representation DNA sequencing (RRS) protocols (e.g., RADseq, ddRAD) have enabled the analysis of genetic data on an unprecedented scale. Here, we present a novel approach for the discovery and statistical validation of sex‐specific loci in GBS data sets. We used GBS to genotype 166 New Zealand fur seals (NZFS, Arctocephalus forsteri) of known sex. We retained monomorphic loci as potential sex‐specific markers in the locus discovery phase. We then used (i) a sex‐specific locus threshold (SSLT) to identify significantly male‐specific loci within our data set; and (ii) a significant sex‐assignment threshold (SSAT) to confidently assign sex in silico the presence or absence of significantly male‐specific loci to individuals in our data set treated as unknowns (98.9% accuracy for females; 95.8% for males, estimated via cross‐validation). Furthermore, we assigned sex to 86 individuals of true unknown sex using our SSAT and assessed the effect of SSLT adjustments on these assignments. From 90 verified sex‐specific loci, we developed a panel of three sex‐specific PCR primers that we used to ascertain sex independently of our GBS data, which we show amplify reliably in at least two other pinniped species. Using monomorphic loci normally discarded from large SNP data sets is an effective way to identify robust sex‐linked markers for nonmodel species. Our novel pipeline can be used to identify and statistically validate monomorphic and polymorphic sex‐specific markers across a range of species and RRS data sets.