Next-generation DNA Sequencing Methods Research Articles

Genomic Divergence and the Evolution of Ecotypes in Bottlenose Dolphins (Genus Tursiops).

Climatic changes have caused major environmental restructuring throughout the world's oceans. Marine organisms have responded to novel conditions through various biological systems, including genomic adaptation. Growing accessibility of next-generation DNA sequencing methods to study nonmodel species has recently allowed genomic changes underlying environmental adaptations to be investigated. This study used double-digest restriction-site associated DNA (ddRAD) sequence data to investigate the genomic basis of ecotype formation across currently recognized species and subspecies of bottlenose dolphins (genus Tursiops) in the Southern Hemisphere. Subspecies-level genomic divergence was confirmed between the offshore common bottlenose dolphin (T. truncatus truncatus) and the inshore Lahille's bottlenose dolphin (T. t. gephyreus) from the southwestern Atlantic Ocean (SWAO). Similarly, subspecies-level divergence is suggested between inshore (eastern Australia) Indo-Pacific bottlenose dolphin (T. aduncus) and the proposed Burrunan dolphin (T. australis) from southern Australia. Inshore bottlenose dolphin lineages generally had lower genomic diversity than offshore lineages, a pattern particularly evident for T. t. gephyreus, which showed exceptionally low diversity. Genomic regions associated with cardiovascular, musculoskeletal, and energy production systems appear to have undergone repeated adaptive evolution in inshore lineages across the Southern Hemisphere. We hypothesize that comparable selective pressures in the inshore environment drove similar adaptive responses in each lineage, supporting parallel evolution of inshore bottlenose dolphins. With climate change altering marine ecosystems worldwide, it is crucial to gain an understanding of the adaptive capacity of local species and populations. Our study provides insights into key adaptive pathways that may be important for the long-term survival of cetaceans and other organisms in a changing marine environment.

Effects of Active Volcanism on Bacterial Communities in the Highest-Altitude Crater Lake of Ojos del Salado (Dry Andes, Altiplano-Atacama Region).

Periglacial and volcanic environments are considered terrestrial analogs of Mars with regard to astrobiological characteristics due to their specific set of extreme features. Ojos del Salado, the highest volcano on Earth (6893 m a.s.l.), is surrounded by several craters, one of which harbors the highest known altitude lake (6480 m a.s.l.), which is influenced by a rare combination of extreme environmental factors, that is, low mean temperature, permafrost, fumarolic activity, acidity, and extreme low organic matter content. To assess the genetic diversity and ecological tolerance of bacteria, samples were taken in February 2016 from the sediments covered with acidic cold (pH 4.88, 3.8°C) and warm (pH 2.08, 40.8°C) water. As a control, a nonvolcanic high-altitude lake (at 5900 m a.s.l.) was also studied by both cultivation-based and next-generation DNA sequencing methods. Isolates from the crater lake showed tolerance toward acidic pH values, unlike isolates from the nonvolcanic lake. Illumina MiSeq sequencing of the 16S rRNA gene exposed simplified, although characteristically different, bacterial communities in the warm and cold water-saturated sediments. In the fumarolic creek sediments, acidophilic iron oxidizers (Ferrithrix, Gallionella) and iron reducers (Acidiphilium) were abundant, and bacteria involved in the sulfur oxidation (Hydrogenobaculum, Thiomonas) and reduction (Desulfosporosinus) were also detected. Therefore, we propose an integrated model that addresses the potential role of bacteria in the sulfur and iron geomicrobiological cycles.

Usefulness of next-generation DNA sequencing for the diagnosis of urinary tract infection.

Acute urinary tract infection (UTI) is a highly common clinical condition. Although bacterial culture is the gold standard diagnostic test, false negative results may be possible, leading to the pathogen being unidentified. In recent years, bacterial DNA sequencing analysis has garnered much attention, but clinical studies are rare in Japan. In this study, we assessed the usefulness of next-generation DNA sequencing (NGS) analysis for acute UTI patients. We thus performed an observational, retrospective case series study. Urine and blood samples were collected from ten acute UTI patients, of whom four had also been diagnosed with urosepsis. Seven variable regions of bacterial 16S rRNA genes were amplified by PCR and then sequenced by IonPGM. The identified bacterial species were compared with those identified using the culture tests and the clinical parameters were analyzed. As a result, the NGS method effectively identified predominant culture-positive bacteria in urine samples. The urine NGS also detected several culture-negative species, which have been reported to be potentially pathogenic. Out of four urosepsis cases, three were pathogen-positive in blood NGS results, while two were pathogen-negative in blood culture. In one sepsis case, although blood culture was negative for Escherichia coli, this species was detected by blood NGS. For non-sepsis cases, however, blood NGS, as well as blood culture, was less effective in detecting bacterial signals. In conclusion, NGS is potentially useful for identifying pathogenic bacteria in urine from acute UTI patients but is less applicable in patients who do not meet clinical criteria for sepsis.

Characterization of the complete plastid genome of Psammosilene tunicoides (Caryophyllaceae), an endangered medical herb endemic to south-western China

Psammosilene tunicoides is an endangered medical herb endemic to south-western China. In this study, the complete plastid genome of the species was characterized and assembled using the next-generation DNA sequencing method. The plastid genome is 153,978 bp in length, including a large single copy (LSC) region of 83,981 bp and a small single copy (SSC) region of 17,489 bp, which were separated by a pair of inverted repeat (IR) regions of 26,254 bp. The genome encoded 112 unique genes, including 78 protein-coding genes, four ribosomal RNA genes, and 30 transfer RNA genes. The overall GC content of the whole genome is 36.49%. The phylogenetic analysis based on 17 plastid genome of Caryophyllaceae revealed that P. tunicoides nested within the tribe Caryophylleae with strong support value.

Microbial community profiling of peripheral blood in myalgic encephalomyelitis/chronic fatigue syndrome

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is estimated to afflict hundreds of thousands, if not millions, of Americans with vastly more impacted individuals worldwide; however, the etiology of this disease has not been well established. Based on the features of ME/CFS, we hypothesized an unrecognized vascular infection may be involved. To evaluate this possibility, we performed a blinded pilot study of 30 ME/CFS patients meeting the Fukuda criteria and 48 normal controls. A community-wide analysis using next-generation DNA sequencing methods detected prokaryotic and eukaryotic populations in the peripheral blood of both ME/CFS patients and normal controls. Analysis of the prokaryotic portion of the samples revealed that organisms belonging to the Pseudomonas asplenii species, Pseudomonadaceae family, Pseudomonadales order, and γ-proteobacteria class are inversely correlated with RAND-36 scores, a quality of life metric that is reduced in ME/CFS patients. In addition, analysis of the detected eukaryotic species suggests that the Perkinsus genus is also inversely associated with RAND-36 scores. The most frequently observed eukaryotic DNA was for Funneliformis mosseae, an arbuscular mycorrhizal fungus, which was in both ME/CFS and normal control samples. A multivariate composite score consisting of the Perkinsus genus, Spumella genus, and β-proteobacteria class displays an inverse relationship to RAND-36 scores. Lastly, the combined measurements of several taxa allow for a retrospective categorical sorting of ME/CFS patients from normal control samples. These results suggest that microbial DNA signatures, including those from poorly understood eukaryotes, may be differentially detectable in ME/CFS and normal control samples.

Revisiting Criteria for Plant MicroRNA Annotation in the Era of Big Data.

MicroRNAs (miRNAs) are ∼21-nucleotide-long regulatory RNAs that arise from endonucleolytic processing of hairpin precursors. Many function as essential posttranscriptional regulators of target mRNAs and long noncoding RNAs. Alongside miRNAs, plants also produce large numbers of short interfering RNAs (siRNAs), which are distinguished from miRNAs primarily by their biogenesis (typically processed from long double-stranded RNA instead of single-stranded hairpins) and functions (typically via roles in transcriptional regulation instead of posttranscriptional regulation). Next-generation DNA sequencing methods have yielded extensive data sets of plant small RNAs, resulting in many miRNA annotations. However, it has become clear that many miRNA annotations are questionable. The sheer number of endogenous siRNAs compared with miRNAs has been a major factor in the erroneous annotation of siRNAs as miRNAs. Here, we provide updated criteria for the confident annotation of plant miRNAs, suitable for the era of "big data" from DNA sequencing. The updated criteria emphasize replication and the minimization of false positives, and they require next-generation sequencing of small RNAs. We argue that improved annotation systems are needed for miRNAs and all other classes of plant small RNAs. Finally, to illustrate the complexities of miRNA and siRNA annotation, we review the evolution and functions of miRNAs and siRNAs in plants.

Genomic Characterization of Dairy Associated Leuconostoc Species and Diversity of Leuconostocs in Undefined Mixed Mesophilic Starter Cultures.

Undefined mesophilic mixed (DL-type) starter cultures are composed of predominantly Lactococcus lactis subspecies and 1–10% Leuconostoc spp. The composition of the Leuconostoc population in the starter culture ultimately affects the characteristics and the quality of the final product. The scientific basis for the taxonomy of dairy relevant leuconostocs can be traced back 50 years, and no documentation on the genomic diversity of leuconostocs in starter cultures exists. We present data on the Leuconostoc population in five DL-type starter cultures commonly used by the dairy industry. The analyses were performed using traditional cultivation methods, and further augmented by next-generation DNA sequencing methods. Bacterial counts for starter cultures cultivated on two different media, MRS and MPCA, revealed large differences in the relative abundance of leuconostocs. Most of the leuconostocs in two of the starter cultures were unable to grow on MRS, emphasizing the limitations of culture-based methods and the importance of careful media selection or use of culture independent methods. Pan-genomic analysis of 59 Leuconostoc genomes enabled differentiation into twelve robust lineages. The genomic analyses show that the dairy-associated leuconostocs are highly adapted to their environment, characterized by the acquisition of genotype traits, such as the ability to metabolize citrate. In particular, Leuconostoc mesenteroides subsp. cremoris display telltale signs of a degenerative evolution, likely resulting from a long period of growth in milk in association with lactococci. Great differences in the metabolic potential between Leuconostoc species and subspecies were revealed. Using targeted amplicon sequencing, the composition of the Leuconostoc population in the five commercial starter cultures was shown to be significantly different. Three of the cultures were dominated by Ln. mesenteroides subspecies cremoris. Leuconostoc pseudomesenteroides dominated in two of the cultures while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only present in low amounts in one of the cultures. This is the first in-depth study of Leuconostoc genomics and diversity in dairy starter cultures. The results and the techniques presented may be of great value for the dairy industry.

De Novo Assembly and Genome Analyses of the Marine-Derived Scopulariopsis brevicaulis Strain LF580 Unravels Life-Style Traits and Anticancerous Scopularide Biosynthetic Gene Cluster.

The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis.

Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter-Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method.

It can be useful to know the transgene insertion site in transgenic mice for a variety of reasons, but determining the insertion site generally is a time consuming, expensive, and laborious task. A simple method is presented to determine transgene insertion sites that combines the enrichment of a sequencing library by polymerase chain reaction (PCR) for sequences containing the transgene, followed by next-generation sequencing of the enriched library. This method was applied to determine the site of integration of the thyroid peroxidase promoter-Cre recombinase mouse transgene that is commonly used to create thyroid-specific gene deletions. The insertion site was found to be between bp 12,372,316 and 12,372,324 on mouse chromosome 9, with the nearest characterized genes being Cntn5 and Jrkl, ∼1.5 and 0.9 Mbp from the transgene, respectively. One advantage of knowing a transgene insertion site is that it facilitates distinguishing hemizygous from homozygous transgenic mice. Although this can be accomplished by real-time quantitative PCR, the expected Ct difference is only one cycle, which is challenging to assess accurately. Therefore, the transgene insertion site information was used to develop a 3-primer qualitative PCR assay that readily distinguishes wild type, hemizygous, and homozygous TPO-Cre mice based upon size differences of the wild type and transgenic allele PCR products. Identification of the genomic insertion site of the thyroid peroxidase promoter-Cre mouse transgene should facilitate the use of these mice in studies of thyroid biology.

Mutation in CPT1C Associated With Pure Autosomal Dominant Spastic Paraplegia.

The family of genes implicated in hereditary spastic paraplegias (HSPs) is quickly expanding, mostly owing to the widespread availability of next-generation DNA sequencing methods. Nevertheless, a genetic diagnosis remains unavailable for many patients. To identify the genetic cause for a novel form of pure autosomal dominant HSP. We examined and followed up with a family presenting to a tertiary referral center for evaluation of HSP for a decade until August 2014. Whole-exome sequencing was performed in 4 patients from the same family and was integrated with linkage analysis. Sanger sequencing was used to confirm the presence of the candidate variant in the remaining affected and unaffected members of the family and screen the additional patients with HSP. Five affected and 6 unaffected participants from a 3-generation family with pure adult-onset autosomal dominant HSP of unknown genetic origin were included. Additionally, 163 unrelated participants with pure HSP of unknown genetic cause were screened. Mutation in the neuronal isoform of carnitine palmitoyl-transferase (CPT1C) gene. We identified the nucleotide substitution c.109C>T in exon 3 of CPT1C, which determined the base substitution of an evolutionarily conserved Cys residue for an Arg in the gene product. This variant strictly cosegregated with the disease phenotype and was absent in online single-nucleotide polymorphism databases and in 712 additional exomes of control participants. We showed that CPT1C, which localizes to the endoplasmic reticulum, is expressed in motor neurons and interacts with atlastin-1, an endoplasmic reticulum protein encoded by the ATL1 gene known to be mutated in pure HSPs. The mutation, as indicated by nuclear magnetic resonance spectroscopy studies, alters the protein conformation and reduces the mean (SD) number (213.0 [46.99] vs 81.9 [14.2]; P < .01) and size (0.29 [0.01] vs 0.26 [0.01]; P < .05) of lipid droplets on overexpression in cells. We also observed a reduction of mean (SD) lipid droplets in primary cortical neurons isolated from Cpt1c-/- mice as compared with wild-type mice (1.0 [0.12] vs 0.44 [0.05]; P < .001), suggesting a dominant negative mechanism for the mutation. This study expands the genetics of autosomal dominant HSP and is the first, to our knowledge, to link mutation in CPT1C with a human disease. The association of the CPT1C mutation with changes in lipid droplet biogenesis supports a role for altered lipid-mediated signal transduction in HSP pathogenesis.

Next-generation DNA sequencing reveals that low fungal diversity in house dust is associated with childhood asthma development.

Dampness and visible mold in homes are associated with asthma development, but causal mechanisms remain unclear. The goal of this research was to explore associations among measured dampness, fungal exposure, and childhood asthma development without the bias of culture-based microbial analysis. In the low-income, Latino CHAMACOS birth cohort, house dust was collected at age 12 months, and asthma status was determined at age 7 years.The current analysis included 13 asthma cases and 28 controls. Next-generation DNA sequencing methods quantified fungal taxa and diversity. Lower fungal diversity (number of fungal operational taxonomic units) was significantly associated with increased risk of asthma development: unadjusted odds ratio(OR) 4.80 (95% confidence interval (CI) 1.04–22.1). Control for potential confounders strengthened this relationship. Decreased diversity within the genus Cryptococcus was significantly associated with increased asthma risk (OR 21.0, 95% CI 2.16–204). No fungal taxon (species, genus, class) was significantly positively associated with asthma development, and one was significantly negatively associated. Elevated moisture was associated with increased fungal diversity, and moisture/mold indicators were associated with four fungal taxa. Next-generation DNA sequencing provided comprehensive estimates of fungal identity and diversity, demonstrating significant associations between low fungal diversity and childhood asthma development in this community. Early life exposure to low fungal diversity in house dust was associated with increased risk for later asthma developmen tin this low-income, immigrant community. No individual fungal taxon (species, genus, or class) was associated with asthma development, although exposure to low diversity within the genus Cryptococcus was associated with asthma development. Future asthma development studies should incorporate fungal diversity measurements, in addition to measuring individual fungal taxa. These results represent a step toward identifying the aspect(s) of indoor microbial populations that are associated with asthma development and suggest that understanding the factors that control diversity in the indoor environment may lead to public health recommendations for asthma prevention in the future.

Below-ground fine-scale distribution and soil versus fine root detection of fungal and soil oomycete communities in a French beech forest

The use of next-generation DNA sequencing methods, which produce massive volumes of data, has transformed fungal molecular ecology. These technologies offer the opportunity to describe microbial communities in depth and to investigate fungal diversity at a large geographical scale. In particular, due to the emergence of studies conducted at a large scale (countries, continents), the definition of a compromise between the optimal and representative description of local diversity (plot scale) and the management of a greater number of sites for these studies is required. This work was performed to explore the local richness and the structure of the ectomycorrhizal (EcM) fungal and oomycete communities in a temperate beech plot using 454 pyrosequencing technology. The internal transcribed spacer 1 (ITS1) region was amplified and sequenced from fine roots and soil sampled from the two upper horizons. Our analyses suggest that soil could be a good substitute for fine roots in studying EcM fungi. The fungal assemblage exhibited a vertical distribution in the soil profile. Only four Pythiaceae were identified, which was insufficient to study the spatial distribution of this group. The fungal and particularly the ectomycorrhizal (EcM) richness was not correlated with any soil variable (pH, C, N or P), but the spatial structure was explained by the C:N ratio and phosphorus content. Our data reveal the importance of maintaining a minimal representative sampling at the plot scale for comparative geographical studies.

Human Microbiome in Health and Disease

Mammals are complex assemblages of mammalian and bacterial cells organized into functional organs, tissues, and cellular communities. Human biology can no longer concern itself only with human cells: Microbiomes at different body sites and functional metagenomics must be considered part of systems biology. The emergence of metagenomics has resulted in the generation of vast data sets of microbial genes and pathways present in different body habitats. The profound differences between microbiomes in various body sites reveal how metagenomes contribute to tissue and organ function. As next-generation DNA-sequencing methods provide whole-metagenome data in addition to gene-expression profiling, metaproteomics, and metabonomics, differences in microbial composition and function are being linked to health and disease states in different organs and tissues. Global parameters of microbial communities may provide valuable information regarding human health status and disease predisposition. More detailed knowledge of the human microbiome will yield next-generation diagnostics and therapeutics for various acute, chronic, localized, and systemic human diseases.

Understanding the pathogenesis of Alzheimer’s disease: will RNA‐Seq realize the promise of transcriptomics?

The prevalence of Alzheimer's disease (AD) is increasing rapidly in the western world and is poised to have a significant economic and societal impact. Current treatments do not alter the underlying disease processes meaning new treatments are required if this imminent epidemic is to be averted. The clinical manifestations of AD are secondary to a substantial loss of cortical neurons. To be effective, neuroprotective strategies will need to be implemented prior to this cell loss. However, this requires the discovery of both pre-clinical markers to identify susceptible patients and the early pathogenic mechanisms to serve as therapeutic targets. Although the biomarkers and pathogenic mechanisms may overlap, it is likely that new approaches are required to identify novel elements of the disease. Transcriptomic analyses, that assume no a priori etiological hypotheses, promise much in elucidating the pathogenesis of complex diseases like AD. Microarrays are the most popular platform for transcriptomic analysis and have been applied across AD models, patient samples and postmortem brain tissue. The results of these studies have been largely discordant which could, to some extent, reflect the limitations of this probe-hybridization-based methodology. In comparison, whole transcriptome sequencing (RNA-Seq) utilizes a highly efficient, next-generation DNA sequencing method with improved dynamic range and scope of transcript detection. RNA-Seq is not only highly suited to investigations of the genomically complex human brain tissue but it can potentially overcome technical issues inherent to case-control comparisons of postmortem brain tissue in neurodegenerative diseases. The volume of data generated by this platform looms as the major logistical hurdle and a systematic experimental approach will be required to maximise the detection of pathogenically relevant signals. Nevertheless, RNA-Seq looks set to deliver a quantum leap forward in our understanding of AD pathogenesis.

Analyzing Gene Expression Data from Microarray and Next‐Generation DNA Sequencing Transcriptome Profiling Assays Using GeneSifter Analysis Edition

Transcription profiling with microarrays has become a standard procedure for comparing the levels of gene expression between pairs of samples, or multiple samples following different experimental treatments. New technologies, collectively known as next-generation DNA sequencing methods, are also starting to be used for transcriptome analysis. These technologies, with their low background, large capacity for data collection, and dynamic range, provide a powerful and complementary tool to the assays that formerly relied on microarrays. In this chapter, we describe two protocols for working with microarray data from pairs of samples and samples treated with multiple conditions, and discuss alternative protocols for carrying out similar analyses with next-generation DNA sequencing data from two different instrument platforms (Illumina GA and Applied Biosystems SOLiD).

Perspectives of DNA microarray and next-generation DNA sequencing technologies

DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequencing method was first introduced by the 454 Company in 2003, immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research.