Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues

The increasing use of high‐throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low‐salt CTAB (MoLSC) extraction protocol to accommodate contaminant‐rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in‐house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuitable for some applications of massively parallel sequencing. Our open‐source MoLSC protocol provides a cost‐effective, scalable, alternative DNA extraction method that can be easily optimized and adapted for sequencing applications in other contaminant‐rich samples.     

Mapping‐by‐sequencing in complex polyploid genomes using genic sequence capture: a case study to map yellow rust resistance in hexaploid wheat

Previously we extended the utility of mapping‐by‐sequencing by combining it with sequence capture and mapping sequence data to pseudo‐chromosomes that were organized using wheat–Brachypodium synteny. This, with a bespoke haplotyping algorithm, enabled us to map the flowering time locus in the diploid wheat Triticum monococcum L. identifying a set of deleted genes (Gardiner et al., 2014). Here, we develop this combination of gene enrichment and sliding window mapping‐by‐synteny analysis to map the Yr6 locus for yellow stripe rust resistance in hexaploid wheat. A 110 MB NimbleGen capture probe set was used to enrich and sequence a doubled haploid mapping population of hexaploid wheat derived from an Avalon and Cadenza cross. The Yr6 locus was identified by mapping to the POPSEQ chromosomal pseudomolecules using a bespoke pipeline and algorithm (Chapman et al., 2015). Furthermore the same locus was identified using newly developed pseudo‐chromosome sequences as a mapping reference that are based on the genic sequence used for sequence enrichment. The pseudo‐chromosomes allow us to demonstrate the application of mapping‐by‐sequencing to even poorly defined polyploidy genomes where chromosomes are incomplete and sub‐genome assemblies are collapsed. This analysis uniquely enabled us to: compare wheat genome annotations; identify the Yr6 locus – defining a smaller genic region than was previously possible; associate the interval with one wheat sub‐genome and increase the density of SNP markers associated. Finally, we built the pipeline in iPlant, making it a user‐friendly community resource for phenotype mapping.     

Identification of a large SNP dataset in Larimichthys crocea using specific‐locus amplified fragment sequencing

The large yellow croaker, Larimichthys crocea, is a commercially important drum fish (Family: Sciaenidae) native to the East and South China Sea. Habitat deterioration and overfishing have led to significant population decline and the collapse of its fishery over the past decades. Today, the market supply of L. crocea depends solely on stocks produced in hatcheries and farms. Common issues that occur in the culture of L. crocea include germplasm degradation, precocious puberty, elevated disease susceptibility and growth retardation. In this study, we employed SLAF‐seq (specific‐locus amplified fragment sequencing) technology to identify single nucleotide polymorphism (SNP) loci across the L. crocea genome. Sixty samples were selected for SLAF analysis out of 1000 progeny in the same cohort of a cultured stock. Our analysis obtained a total of 151 253 SLAFs, of which 65.88% (99 652) were identified to be polymorphic, scoring a total of 710 567 putative SNPs. Further filtration resulted in a final panel of 1782 SNP loci. The data derived from this work could be beneficial for understanding the genetics of complex phenotypic traits as well as for developing marker‐selection‐assisted breeding programs in L. crocea.     

Genotyping‐by‐sequencing for estimating relatedness in nonmodel organisms: Avoiding the trap of precise bias

There has been remarkably little attention to using the high resolution provided by genotyping‐by‐sequencing (i.e., RADseq and similar methods) for assessing relatedness in wildlife populations. A major hurdle is the genotyping error, especially allelic dropout, often found in this type of data that could lead to downward‐biased, yet precise, estimates of relatedness. Here, we assess the applicability of genotyping‐by‐sequencing for relatedness inferences given its relatively high genotyping error rate. Individuals of known relatedness were simulated under genotyping error, allelic dropout and missing data scenarios based on an empirical ddRAD data set, and their true relatedness was compared to that estimated by seven relatedness estimators. We found that an estimator chosen through such analyses can circumvent the influence of genotyping error, with the estimator of Ritland (Genetics Research, 67, 175) shown to be unaffected by allelic dropout and to be the most accurate when there is genotyping error. We also found that the choice of estimator should not rely solely on the strength of correlation between estimated and true relatedness as a strong correlation does not necessarily mean estimates are close to true relatedness. We also demonstrated how even a large SNP data set with genotyping error (allelic dropout or otherwise) or missing data still performs better than a perfectly genotyped microsatellite data set of tens of markers. The simulation‐based approach used here can be easily implemented by others on their own genotyping‐by‐sequencing data sets to confirm the most appropriate and powerful estimator for their data.     

Chromosomal‐level assembly of Takifugu obscurus (Abe, 1949) genome using third‐generation DNA sequencing and Hi‐C analysis

The Tetraodontidae family are known to have relatively small and compact genomes compared to other vertebrates. The obscure puffer fish Takifugu obscurus is an anadromous species that migrates to freshwater from the sea for spawning. Thus the euryhaline characteristics of T. obscurus have been investigated to gain understanding of their survival ability, osmoregulation, and other homeostatic mechanisms in both freshwater and seawater. In this study, a high quality chromosome‐level reference genome for T. obscurus was constructed using long‐read Pacific Biosciences (PacBio) Sequel sequencing and a Hi‐C‐based chromatin contact map platform. The final genome assembly of T. obscurus is 381 Mb, with a contig N50 length of 3,296 kb and longest length of 10.7 Mb, from a total of 62 Gb of raw reads generated using single‐molecule real‐time sequencing technology from a PacBio Sequel platform. The PacBio data were further clustered into chromosome‐scale scaffolds using a Hi‐C approach, resulting in a 373 Mb genome assembly with a contig N50 length of 15.2 Mb and and longest length of 28 Mb. When we directly compared the 22 longest scaffolds of T. obscurus to the 22 chromosomes of the tiger puffer Takifugu rubripes, a clear one‐to‐one orthologous relationship was observed between the two species, supporting the chromosome‐level assembly of T. obscurus. This genome assembly can serve as a valuable genetic resource for exploring fugu‐specific compact genome characteristics, and will provide essential genomic information for understanding molecular adaptations to salinity fluctuations and the evolution of osmoregulatory mechanisms.     

Batch effects in a multiyear sequencing study: False biological trends due to changes in read lengths

High‐throughput sequencing is a powerful tool, but suffers biases and errors that must be accounted for to prevent false biological conclusions. Such errors include batch effects; technical errors only present in subsets of data due to procedural changes within a study. If overlooked and multiple batches of data are combined, spurious biological signals can arise, particularly if batches of data are correlated with biological variables. Batch effects can be minimized through randomization of sample groups across batches. However, in long‐term or multiyear studies where data are added incrementally, full randomization is impossible, and batch effects may be a common feature. Here, we present a case study where false signals of selection were detected due to a batch effect in a multiyear study of Alpine ibex (Capra ibex). The batch effect arose because sequencing read length changed over the course of the project and populations were added incrementally to the study, resulting in nonrandom distributions of populations across read lengths. The differences in read length caused small misalignments in a subset of the data, leading to false variant alleles and thus false SNPs. Pronounced allele frequency differences between populations arose at these SNPs because of the correlation between read length and population. This created highly statistically significant, but biologically spurious, signals of selection and false associations between allele frequencies and the environment. We highlight the risk of batch effects and discuss strategies to reduce the impacts of batch effects in multiyear high‐throughput sequencing studies.     

Advancing biodiversity assessments with environmental DNA: Long‐read technologies help reveal the drivers of Amazonian fungal diversity

Fungi are a key component of tropical biodiversity. However, due to their inconspicuous and largely subterranean nature, they are usually neglected in biodiversity inventories. The goal of this study was to identify the key determinants of fungal richness, community composition, and turnover in tropical rainforests. We tested specifically for the effect of soil properties, habitat, and locality in Amazonia. For these analyses, we used high‐throughput sequencing data of short and long reads of fungal DNA present in soil and organic litter samples, combining existing and novel genomic data. Habitat type (phytophysiognomy) emerges as the strongest factor explaining fungal community composition. Naturally open areas—campinas—are the richest habitat overall. Soil properties have different effects depending on the soil layer (litter or mineral soil) and the choice of genetic marker. We suggest that campinas could be a neglected hotspot of fungal diversity. An underlying cause for their rich diversity may be the overall low soil fertility, which increases the reliance on biotic interactions essential for nutrient absorption in these environments, notably ectomycorrhizal fungi–plant associations. Our results highlight the advantages of using both short and long DNA reads produced through high‐throughput sequencing to characterize fungal diversity. While short reads can suffice for diversity and community comparison, long reads add taxonomic precision and have the potential to reveal population diversity.     

Next‐generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms

The recent emergence of barcoding approaches coupled to those of next‐generation sequencing (NGS) has raised new perspectives for studying environmental communities. In this framework, we tested the possibility to derive accurate inventories of diatom communities from pyrosequencing outputs with an available DNA reference library. We used three molecular markers targeting the nuclear, chloroplast and mitochondrial genomes (SSU rDNA, rbcL and cox1) and three samples of a mock community composed of 30 known diatom strains belonging to 21 species. In the goal to detect methodological biases, one sample was constituted directly from pooled cultures, whereas the others consisted of pooled PCR products. The NGS reads obtained by pyrosequencing (Roche 454) were compared first to a DNA reference library including the sequences of all the species used to constitute the mock community, and second to a complete DNA reference library with a larger taxonomic coverage. A stringent taxonomic assignation gave inventories that were compared to the real one. We detected biases due to DNA extraction and PCR amplification that resulted in false‐negative detection. Conversely, pyrosequencing errors appeared to generate false positives, especially in case of closely allied species. The taxonomic coverage of DNA reference libraries appears to be the most crucial factor, together with marker polymorphism which is essential to identify taxa at the species level. RbcL offers a high resolving power together with a large DNA reference library. Although needing further optimization, pyrosequencing is suitable for identifying diatom assemblages and may find applications in the field of freshwater biomonitoring.     

Sequence capture using PCR‐generated probes: a cost‐effective method of targeted high‐throughput sequencing for nonmodel organisms

Recent advances in high‐throughput sequencing library preparation and subgenomic enrichment methods have opened new avenues for population genetics and phylogenetics of nonmodel organisms. To multiplex large numbers of indexed samples while sequencing predominantly orthologous, targeted regions of the genome, we propose modifications to an existing, in‐solution capture that utilizes PCR products as target probes to enrich library pools for the genomic subset of interest. The sequence capture using PCR‐generated probes (SCPP) protocol requires no specialized equipment, is highly flexible and significantly reduces experimental costs for projects where a modest scale of genetic data is optimal (25–100 genomic loci). Our alterations enable application of this method across a wider phylogenetic range of taxa and result in higher capture efficiencies and coverage at each locus. Efficient and consistent capture over multiple SCPP experiments and at various phylogenetic distances is demonstrated, extending the utility of this method to both phylogeographic and phylogenomic studies.     

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.     

Modular tagging of amplicons using a single PCR for high‐throughput sequencing

High‐throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate‐limiting step. For example, HTS platforms require platform‐specific adapter sequences to be present at the 5′ and 3′ end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean‐up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost‐effective and efficient method to prepare amplicons for HTS.     

Elucidating cryptic dynamics of Theileria communities in African buffalo using a high‐throughput sequencing informatics approach

1. Increasing access to next‐generation sequencing (NGS) technologies is revolutionizing the life sciences. In disease ecology, NGS‐based methods have the potential to provide higher‐resolution data on communities of parasites found in individual hosts as well as host populations.
2. Here, we demonstrate how a novel analytical method, utilizing high‐throughput sequencing of PCR amplicons, can be used to explore variation in blood‐borne parasite (Theileria—Apicomplexa: Piroplasmida) communities of African buffalo at higher resolutions than has been obtained with conventional molecular tools.
3. Results reveal temporal patterns of synchronized and opposite fluctuations of prevalence and relative abundance of Theileria spp. within the host population, suggesting heterogeneous transmission across taxa. Furthermore, we show that the community composition of Theileria spp. and their subtypes varies considerably between buffalo, with differences in composition reflected in mean and variance of overall parasitemia, thereby showing potential to elucidate previously unexplained contrasts in infection outcomes for host individuals.
4. Importantly, our methods are generalizable as they can be utilized to describe blood‐borne parasite communities in any host species. Furthermore, our methodological framework can be adapted to any parasite system given the appropriate genetic marker.
5. The findings of this study demonstrate how a novel NGS‐based analytical approach can provide fine‐scale, quantitative data, unlocking opportunities for discovery in disease ecology.

     

Land use is a determinant of plant pathogen alpha‐ but not beta‐diversity

Little is known about the diversity patterns of plant pathogens and how they change with land use at a broad scale. We employed DNA metabarcoding to describe the diversity and composition of putative plant pathogen communities in three substrates (soil, roots, and leaves) across five major land uses at a national scale. Almost all plant pathogen communities (fungi, oomycetes, and bacteria) showed strong responses to land use and substrate type. Land use category could explain up to 24% of the variance in composition between communities. Alpha‐diversity (richness) of plant pathogens was consistently lower in natural forests than in agricultural systems. In planted forests, there was also generally low pathogen alpha‐diversity in soil and roots, but alpha‐diversity in leaves was high compared with most other land uses. In contrast to alpha‐diversity, differences in within‐land use beta‐diversity of plant pathogens (the predictability of plant pathogen communities within land use) were subtle. Our results show that large‐scale patterns and distributions of putative plant pathogens can be determined using metabarcoding, allowing some of the first landscape level insights into these critically important communities.     

Dietary n‐3 long‐chain polyunsaturated fatty acids modify phosphoenolpyruvate carboxykinase activity and lipid synthesis from glucose in adipose tissue of rats fed a high‐sucrose diet

Long‐chain polyunsaturated n‐3 fatty acids (n‐3 LCPUFAs) have hypolipidemic effects and modulate intermediary metabolism to prevent or reverse insulin resistance in a way that is not completely elucidated. Here, effects of these fatty acids on the lipid profile, phosphoenolpyruvate carboxykinase (PEPCK) activity, lipid synthesis from glucose in epididymal adipose tissue (Ep‐AT) and liver were investigated. Male rats were fed a high‐sucrose diet (SU diet), containing either sunflower oil or a mixture of sunflower and fish oil (SU–FO diet), and the control group was fed a standard diet. After 13 weeks, liver, adipose tissue and blood were harvested and analysed. The dietary n‐3 LCPUFAs prevented sucrose‐induced increase in adiposity and serum free fat acids, serum and hepatic triacylglycerol and insulin levels. Furthermore, these n‐3 LCPUFAs decreased lipid synthesis from glucose and increased PEPCK activity in the Ep‐AT of rats fed the SU–FO diet compared to those fed the SU diet, besides reducing lipid synthesis from glucose in hepatic tissue. Thus, the inclusion of n‐3 LCPUFAs in the diet may be beneficial for the prevention or attenuation of dyslipidemia and insulin resistance, and for reducing the risk of related chronic diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

Paralogs are revealed by proportion of heterozygotes and deviations in read ratios in genotyping‐by‐sequencing data from natural populations

Whole‐genome duplications have occurred in the recent ancestors of many plants, fish, and amphibians, resulting in a pervasiveness of paralogous loci and the potential for both disomic and tetrasomic inheritance in the same genome. Paralogs can be difficult to reliably genotype and are often excluded from genotyping‐by‐sequencing (GBS) analyses; however, removal requires paralogs to be identified which is difficult without a reference genome. We present a method for identifying paralogs in natural populations by combining two properties of duplicated loci: (i) the expected frequency of heterozygotes exceeds that for singleton loci, and (ii) within heterozygotes, observed read ratios for each allele in GBS data will deviate from the 1:1 expected for singleton (diploid) loci. These deviations are often not apparent within individuals, particularly when sequence coverage is low; but, we postulated that summing allele reads for each locus over all heterozygous individuals in a population would provide sufficient power to detect deviations at those loci. We identified paralogous loci in three species: Chinook salmon (Oncorhynchus tshawytscha) which retains regions with ongoing residual tetrasomy on eight chromosome arms following a recent whole‐genome duplication, mountain barberry (Berberis alpina) which has a large proportion of paralogs that arose through an unknown mechanism, and dusky parrotfish (Scarus niger) which has largely rediploidized following an ancient whole‐genome duplication. Importantly, this approach only requires the genotype and allele‐specific read counts for each individual, information which is readily obtained from most GBS analysis pipelines.     

The decline of the Turtle Dove: Dietary associations with body condition and competition with other columbids analysed using high‐throughput sequencing

Dietary changes linked to the availability of anthropogenic food resources can have complex implications for species and ecosystems, especially when species are in decline. Here, we use recently developed primers targeting the ITS2 region of plants to characterize diet from faecal samples of four UK columbids, with particular focus on the European turtle dove (Streptopelia turtur), a rapidly declining obligate granivore. We examine dietary overlap between species (potential competition), associations with body condition in turtle doves and spatiotemporal variation in diet. We identified 143 taxonomic units, of which we classified 55% to species, another 34% to genus and the remaining 11% to family. We found significant dietary overlap between all columbid species, with the highest between turtle doves and stock doves (Columba oenas), then between turtle doves and woodpigeons (Columba palumbus). The lowest overlap was between woodpigeons and collared doves (Streptopelia decaocto). We show considerable change in columbid diets compared to previous studies, probably reflecting opportunistic foraging behaviour by columbids within a highly anthropogenically modified landscape, although our data for nonturtle doves should be considered preliminary. Nestling turtle doves in better condition had a higher dietary proportion of taxonomic units from natural arable plant species and a lower proportion of taxonomic units from anthropogenic food resources such as garden bird seed mixes and brassicas. This suggests that breeding ground conservation strategies for turtle doves should include provision of anthropogenic seeds for adults early in the breeding season, coupled with habitat rich in accessible seeds from arable plants once chicks have hatched.