DNA degradation in fish: Practical solutions and guidelines to improve DNA preservation for genomic research

The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications.     

Prevention,diagnosis and treatment of high‐throughput sequencing data pathologies

High‐throughput sequencing (HTS) technologies generate millions of sequence reads from DNA/RNA molecules rapidly and cost‐effectively, enabling single investigator laboratories to address a variety of ‘omics’ questions in nonmodel organisms, fundamentally changing the way genomic approaches are used to advance biological research. One major challenge posed by HTS is the complexity and difficulty of data quality control (QC). While QC issues associated with sample isolation, library preparation and sequencing are well known and protocols for their handling are widely available, the QC of the actual sequence reads generated by HTS is often overlooked. HTS‐generated sequence reads can contain various errors, biases and artefacts whose identification and amelioration can greatly impact subsequent data analysis. However, a systematic survey on QC procedures for HTS data is still lacking. In this review, we begin by presenting standard ‘health check‐up’ QC procedures recommended for HTS data sets and establishing what ‘healthy’ HTS data look like. We next proceed by classifying errors, biases and artefacts present in HTS data into three major types of ‘pathologies’, discussing their causes and symptoms and illustrating with examples their diagnosis and impact on downstream analyses. We conclude this review by offering examples of successful ‘treatment’ protocols and recommendations on standard practices and treatment options. Notwithstanding the speed with which HTS technologies – and consequently their pathologies – change, we argue that careful QC of HTS data is an important – yet often neglected – aspect of their application in molecular ecology, and lay the groundwork for developing a HTS data QC ‘best practices’ guide.     

Translational genomics for plant breeding with the genome sequence explosion

The use of next‐generation sequencers and advanced genotyping technologies has propelled the field of plant genomics in model crops and plants and enhanced the discovery of hidden bridges between genotypes and phenotypes. The newly generated reference sequences of unstudied minor plants can be annotated by the knowledge of model plants via translational genomics approaches. Here, we reviewed the strategies of translational genomics and suggested perspectives on the current databases of genomic resources and the database structures of translated information on the new genome. As a draft picture of phenotypic annotation, translational genomics on newly sequenced plants will provide valuable assistance for breeders and researchers who are interested in genetic studies.     

Next generation sequencing of Apis mellifera syriaca identifies genes for Varroa resistance and beneficial bee keeping traits

Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next‐generation sequencing (NGS) technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses characteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in‐depth functional annotation methods to identify ～600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK‐STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function‐specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development (Cactus, Relish, dorsal, Ank2, baz), Varroa hygiene (NorpA2, Zasp, LanA, gasp, impl3) and Varroa resistance (Pug, pcmt, elk, elf3‐s10, Dscam2, Dhc64C, gro, futsch) functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.     

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.     

Comparison of random and SSR-enriched shotgun pyrosequencing for microsatellite discovery and single multiplex PCR optimization in Acacia harpophylla F. Muell. Ex Benth

Streamlining the development and genotyping of microsatellites in species for which no genetic information is available represents an important technical challenge to overcome in order to enable mainstream application of state-of-the-art population genetic analysis techniques in nonmodel organisms. Using the example of Acacia harpophylla, an acacia tree endemic of north-eastern Australia, we show that high-throughput shotgun pyrosequencing technology, so-called second-generation sequencing, reduces time and cost of microsatellite marker discovery in nonmodel organisms and of their large-scale typing in natural populations. We found that 0.5% of short sequence reads generated on 454 Genome Sequencer FLX Titanium from random genome sampling and 2.2% of reads generated with prior microsatellite enrichment yielded microsatellite markers with designed polymerase chain reaction (PCR) primers, suggesting that enrichment increases efficiency of pyrosequencing when microsatellite discovery is the primary goal. Using stringent selection criteria to facilitate downstream PCR multiplex design, we identified 1435 microsatellite loci with designed primers from a total of 200,908 short sequence reads. From a subset of 96 loci tested for amplification, 38 were validated for population genetics applications, leading to the optimization of a cost-effective multiplex PCR protocol for the simultaneous typing of nine microsatellites in natural populations of A. harpophylla.     

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.     

Genomic divergence between the migratory and stationary ecotypes of Atlantic cod

Atlantic cod displays a range of phenotypic and genotypic variations, which includes the differentiation into coastal stationary and offshore migratory types of cod that co‐occur in several parts of its distribution range and are often sympatric on the spawning grounds. Differentiation of these ecotypes may involve both historical separation and adaptation to ecologically distinct environments, the genetic basis of which is now beginning to be unravelled. Genomic analyses based on recent sequencing advances are able to document genomic divergence in more detail and may facilitate the exploration of causes and consequences of genome‐wide patterns. We examined genomic divergence between the stationary and migratory types of cod in the Northeast Atlantic, using next‐generation sequencing of pooled DNA from each of two population samples. Sequence data was mapped to the published cod genome sequence, arranged in more than 6000 scaffolds (611 Mb). We identified 25 divergent scaffolds (26 Mb) with a higher than average gene density, against a backdrop of overall moderate genomic differentiation. Previous findings of localized genomic divergence in three linkage groups were confirmed, including a large (15 Mb) genomic region, which seems to be uniquely involved in the divergence of migratory and stationary cod. The results of the pooled sequencing approach support and extend recent findings based on single‐nucleotide polymorphism markers and suggest a high degree of reproductive isolation between stationary and migratory cod in the North‐east Atlantic.     

Genetic and genomic resources of sorghum to connect genotype with phenotype in contrasting environments

With the recent development of genomic resources and high‐throughput phenotyping platforms, the 21st century is primed for major breakthroughs in the discovery, understanding and utilization of plant genetic variation. Significant advances in agriculture remain at the forefront to increase crop production and quality to satisfy the global food demand in a changing climate all while reducing the environmental impacts of the world's food production. Sorghum, a resilient C₄ grain and grass important for food and energy production, is being extensively dissected genetically and phenomically to help connect the relationship between genetic and phenotypic variation. Unlike genetically modified crops such as corn or soybean, sorghum improvement has relied heavily on public research; thus, many of the genetic resources serve a dual purpose for both academic and commercial pursuits. Genetic and genomic resources not only provide the foundation to identify and understand the genes underlying variation, but also serve as novel sources of genetic and phenotypic diversity in plant breeding programs. To better disseminate the collective information of this community, we discuss: (i) the genomic resources of sorghum that are at the disposal of the research community; (ii) the suite of sorghum traits as potential targets for increasing productivity in contrasting environments; and (iii) the prospective approaches and technologies that will help to dissect the genotype–phenotype relationship as well as those that will apply foundational knowledge for sorghum improvement.     

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.     

Different filtering strategies of genotyping‐by‐sequencing data provide complementary resolutions of species boundaries and relationships in a clade of sexually deceptive orchids

Ongoing hybridization and retained ancestral polymorphism in rapidly radiating lineages could mask recent cladogenetic events. This presents a challenge for the application of molecular phylogenetic methods to resolve differences between closely related taxa. We reanalyzed published genotyping‐by‐sequencing (GBS) data to infer the phylogeny of four species within the Ophrys sphegodes complex, a recently radiated clade of orchids. We used different data filtering approaches to detect different signals contained in the dataset generated by GBS and estimated their effects on maximum likelihood trees, global F_ST and bootstrap support values. We obtained a maximum likelihood tree with high bootstrap support, separating the species by using a large dataset based on loci shared by at least 30% of accessions. Bootstrap and F_ST values progressively decreased when filtering for loci shared by a higher number of accessions. However, when filtering more stringently to retain homozygous and organellar loci, we identified two main clades. These clades group individuals independently from their a priori species assignment, but were associated with two organellar haplotype clusters. We infer that a less stringent filtering preferentially selects for rapidly evolving lineage‐specific loci, which might better delimit lineages. In contrast, when using homozygous/organellar DNA loci the signature of a putative hybridization event in the lineage prevails over the most recent phylogenetic signal. These results show that using differing filtering strategies on GBS data could dissect the organellar and nuclear DNA phylogenetic signal and yield novel insights into relationships between closely related species.     

Extending RAD tag analysis to microbial ecology: a comparison between MultiLocus Sequence Typing and 2b‐RAD to investigate Listeria monocytogenes genetic structure

The advent of next‐generation sequencing (NGS) has dramatically changed bacterial typing technologies, increasing our ability to differentiate bacterial isolates. Despite it is now possible to sequence a bacterial genome in a few days and at reasonable costs, most genetic analyses do not require whole‐genome sequencing, which also remains impractical for large population samples due to the cost of individual library preparation and bioinformatics. More traditional sequencing approaches, however, such as MultiLocus Sequence Typing (mlst ) are quite laborious and time‐consuming, especially for large‐scale analyses. In this study, a genotyping approach based on restriction site‐associated (RAD) tag sequencing, 2b‐RAD, was applied to characterize Listeria monocytogenes strains. To verify the feasibility of the method, an in silico analysis was performed on 30 available complete genomes. For the same set of strains, in silico mlst analysis was conducted as well. Subsequently, 2b‐RAD and mlst analyses were experimentally carried out on 58 isolates collected from food samples or food‐processing sites. The obtained results demonstrate that 2b‐RAD predicts mlst types and often provides more detailed information on population structure than mlst . Moreover, the majority of variants differentiating identical sequence type isolates mapped against accessory fragments, thus providing additional information to characterize strains. Although mlst still represents a reliable typing method, large‐scale studies on molecular epidemiology and public health, as well as bacterial phylogenetics, population genetics and biosafety could benefit of a low cost and fast turnaround time approach such as the 2b‐RAD analysis proposed here.     

DNA metabarcoding reveals that 200‐μm‐size‐fractionated filtering is unable to discriminate between planktonic microbial and large eukaryotes

Microeukaryotic plankton (0.2–200 μm) are critical components of aquatic ecosystems and key players in global ecological processes. High‐throughput sequencing is currently revolutionizing their study on an unprecedented scale. However, it is currently unclear whether we can accurately, effectively and quantitatively depict the microeukaryotic plankton communities using traditional size‐fractionated filtering combined with molecular methods. To address this, we analysed the eukaryotic plankton communities both with, and without, prefiltering with a 200 μm pore‐size sieve –by using SSU rDNA‐based high‐throughput sequencing on 16 samples with three replicates in each sample from two subtropical reservoirs sampled from January to October in 2013. We found that ~25% reads were classified as metazoan in both size groups. The species richness, alpha and beta diversity of plankton community and relative abundance of reads in 99.2% eukaryotic OTUs showed no significant changes after prefiltering with a 200 μm pore‐size sieve. We further found that both >0.2 μm and 0.2–200 μm eukaryotic plankton communities, especially the abundant plankton subcommunities, exhibited very similar, and synchronous, spatiotemporal patterns and processes associated with almost identical environmental drivers. The lack of an effect on community structure from prefiltering suggests that environmental DNA from larger metazoa is introduced into the smaller size class. Therefore, size‐fractionated filtering with 200 μm is insufficient to discriminate between the eukaryotic plankton size groups in metabarcoding approaches. Our results also highlight the importance of sequencing depth, and strict quality filtering of reads, when designing studies to characterize microeukaryotic plankton communities.