Next-generation DNA Sequencing Techniques Research Articles

Predicting feed efficiency of Angus steers using the gastrointestinal microbiome

Microbial composition of the gastrointestinal tracts is an important factor affecting the variation in feed efficiency in ruminants. Several studies have investigated the composition of the ruminal and fecal microbiotas, as well as their impacts on feed efficiency and digestion. In addition, next-generation DNA sequencing techniques have allowed us to gain a better understanding of such microbiomes. In this study, the beef cattle microbiome data were analyzed using both a multivariate and a univariate approach and the results were compared. Moreover, a statistical procedure to classify calves in two groups with extreme Residual Feed Intake (RFI) values, using their microbiota profile, was developed. Both fecal and ruminal samples were collected from 63 Angus steers at two different time points for evaluation of their microbiomes: at the beginning and at the end of the feedlot. An additional fecal sample was collected at weaning. A total of 149 and 119 bacterial families (BFs) were retrieved from the ruminal and fecal samples, respectively. A Canonical Discriminant Analysis (CDA) was used to investigate whether BFs were able to distinguish between rumen and fecal samples. A sub-sample of 28 steers was divided in two groups based on their feed efficiency status: positive or negative for RFI. Fecal samples collected at weaning were used to assign the positive and negative RFI animals to their corresponding groups using both Stepwise Discriminant Analysis and CDA. Results revealed that CDA was able to distinguish between rumen and fecal samples. Peptostreptococcaceae was the family most associated with the fecal samples, whereas Prevotellaceae the most associated with the ruminal samples. The CDA using 19 BFs selected from the stepwise was able to correctly assign all animals to the proper RFI groups (negative or positive). Rhizobiaceae was the family most associated with negative RFI, whereas Comamonadacea was the family most linked with positive RFI. The results from this study showed that the multivariate approach can be used to improve microbiome data analysis, as well as to predict feed efficiency in beef cattle using information derived from the fecal microbiome.

A Step toward Amino Acid-Labeled DNA Sequencing: Boosting Transmission Sensitivity of Graphene Nanogap.

Existing obstacles in next-generation DNA sequencing techniques, for instance, high noise, high translocation speed, and configurational fluctuations, call for approaches capable of reaching the goal and accelerating the process of personalized medicine development. The labeling nucleotide approach has the potential to overcome these barriers and boost the recognition sensitivity of a solid-state nanodevice. In this theoretical report, the first-principles density functional theory calculations have been employed to study the role of three different labels, tyrosine (Tyr), aspartic acid (Asp), and arginine (Arg), for labeling DNA nucleotides and study their effect in rapid and controlled DNA sequencing at atomic resolution. Remarkable differences in interaction energy values are noticed in all three cases of differently labeled nucleotides. The zero-bias transmission spectra confirm that proposed labels have the ability to detect the individual nucleotide, amplifying the tunneling current sensitivity by several orders of magnitude. The current-voltage characteristics of Arg-labeled nucleotides are found to be promising for single nucleotide recognition even at a very low bias voltage of 0.1 V.

Chromosomal Microarray Analysis in Turkish Patients with Unexplained Developmental Delay and Intellectual Developmental Disorders.

Aneuploids, copy number variations (CNVs), and single nucleotide variants in specific genes are the main genetic causes of developmental delay (DD) and intellectual disability disorder (IDD). These genetic changes can be detected using chromosome analysis, chromosomal microarray (CMA), and next-generation DNA sequencing techniques. Therefore; In this study, we aimed to investigate the importance of CMA in determining the genomic etiology of unexplained DD and IDD in 123 patients. For 123 patients, chromosome analysis, DNA fragment analysis and microarray were performed. Conventional G-band karyotype analysis from peripheral blood was performed as part of the initial screening tests. FMR1 gene CGG repeat number and methylation analysis were carried out to exclude fragile X syndrome. CMA analysis was performed in 123 unexplained IDD/DD patients with normal karyotypes and fragile X screening, which were evaluated by conventional cytogenetics. Forty-four CNVs were detected in 39 (39/123=31.7%) patients. Twelve CNV variant of unknown significance (VUS) (9.75%) patients and 7 CNV benign (5.69%) patients were reported. In 6 patients, one or more pathogenic CNVs were determined. Therefore, the diagnostic efficiency of CMA was found to be 31.7% (39/123). Today, genetic analysis is still not part of the routine in the evaluation of IDD patients who present to psychiatry clinics. A genetic diagnosis from CMA can eliminate genetic question marks and thus alter the clinical management of patients. Approximately one-third of the positive CMA findings are clinically intervenable. However, the emergence of CNVs as important risk factors for multiple disorders increases the need for individuals with comorbid neurodevelopmental conditions to be the priority where the CMA test is recommended.

Microbiota-Immune Interaction in the Pathogenesis of Gut-Derived Infection.

Gut-derived infection is among the most common complications in patients who underwent severe trauma, serious burn, major surgery, hemorrhagic shock or severe acute pancreatitis (SAP). It could cause sepsis and multiple organ dysfunction syndrome (MODS), which are regarded as a leading cause of mortality in these cases. Gut-derived infection is commonly caused by pathological translocation of intestinal bacteria or endotoxins, resulting from the dysfunction of the gut barrier. In the last decades, the studies regarding to the pathogenesis of gut-derived infection mainly focused on the breakdown of intestinal epithelial tight junction and increased permeability. Limited information is available on the roles of intestinal microbial barrier in the development of gut-derived infection. Recently, advances of next-generation DNA sequencing techniques and its utilization has revolutionized the gut microecology, leading to novel views into the composition of the intestinal microbiota and its connections with multiple diseases. Here, we reviewed the recent progress in the research field of intestinal barrier disruption and gut-derived infection, mainly through the perspectives of the dysbiosis of intestinal microbiota and its interaction with intestinal mucosal immune cells. This review presents novel insights into how the gut microbiota collaborates with mucosal immune cells to involve the development of pathological bacterial translocation. The data might have important implication to better understand the mechanism underlying pathological bacterial translocation, contributing us to develop new strategies for prevention and treatment of gut-derived sepsis.

Identification of new species of Kalloconus and Africonus (Gastropoda, Conidae) from the Cabo Verde Islands through mitochondrial genome comparison

The new species Kalloconus marimaris sp. nov. and Africonus angeluquei sp. nov. are described from the material obtained during the MNCN sampling campaigns at the Cabo Verde Islands. The new species have been characterized by examination of their shell and radular teeth morphology and by the comparison of mitochondrial genomes obtained through next-generation DNA sequencing techniques. Phylogenetic analyses suggest that K. marimaris sp. nov., known from Sal Island, is a sister species to Kalloconus ateralbus, whereas A. angeluquei sp. nov. from Maio Island is a sister species to Africonus josephinae and Africonus borgesi from Boa Vista Island. The estimated genetic distances were consistent with the full species rank for the new taxa described herein.

Microbial diversity in lake–river ecotone of Poyang Lake, China

The ecological environment of lake–river ecotone is complex and changeable, it is an important water purification area and pollution buffer zone, where the microorganisms play an important role in the mediation of material cycle, nutrition transfer, and element transformation. Microbial diversity is sensitive to environmental changes. In this study, 12 water samples from the lake–river ecotone of the Poyang Lake in China were collected to determine microbial gene sequences employing next-generation DNA sequencing techniques, and to evaluate total nitrogen (TN) and total phosphorus (TP) using UV spectrophotometric method. The results showed the major Poyang Lake has the largest microbial population, followed by Yao Lake, Ganjiang River and Raohe River. Based on the Shannon and Simpson Index, major Poyang Lake has the largest biodiversity of microbial communities, followed by Ganjiang River, Yao Lake, and Raohe River. Microbial characteristics vary with the TN and TP concentration, for instance, the nitrifying bacteria were relatively rich in Yao Lake and Ganjiang River ecotone, and the polyphosphate-accumulating organisms (PAO) in Raohe River were richer than those in Ganjiang River. The tag numbers of Euryarchaeota, Acidobacteria, Chlamydiae and Proteobacteria in Ganjiang River were more than in Raohe River, while significant differences were found between the major Poyang Lake and other lake–river ecotone, the operational taxonomic units (OTU) number (removing singletons) of major Poyang Lake have 3047, which was far more than the others. The study indicates that the Poyang Lake serves as a huge microbial gene pool, including a large number of DNA from ammonia oxidizing bacteria to ammonia oxidizing archaea.

The genome-wide DNA sequence specificity of the anti-tumour drug bleomycin in human cells.

The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5'-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5'-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5'-GT*A and 5'- TGT* trinucleotide sequences, and 5'-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5'-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine-pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the -3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.

Single-Read De Novo Sequencing Using Nanopore Mspa

Biological nanopores such as MspA are used in a promising next-generation DNA sequencing technique capable of achieving long, single-molecule reads. In our experiments, current is driven through the ∼1 nm constriction of the protein nanopore MspA. Single molecules of DNA are pulled in by the voltage, and held in place by a DNA polymerase. The polymerase pulls the DNA under load in single-nucleotide steps. The current depends on the bases in the narrowest part of the pore. De novo sequencing accuracy with this method is limited by the inability to distinguish different base sequences that have the same currents, and by unpredictable deviations from forward-stepping behavior by the polymerase. We show that by driving the current through the pore with a varying voltage, we obtain additional information about the base content of the DNA. Varying the voltage allows for the identification and correction of skipping and backstepping behavior, and can discriminate between nucleotide sequences that are indistinguishable when driving the system at a constant voltage. We provide initial results demonstrating that this method greatly improves single read de novo sequencing fidelity.

Understanding the biological responses of nanostructured metals and surfaces

Metals produced by Severe Plastic Deformation (SPD) offer distinct advantages for medical applications such as orthopedic devices, in part because of their nanostructured surfaces. We examine the current theoretical foundations and state of knowledge for nanostructured biomaterials surface optimization within the contexts that apply to bulk nanostructured metals, differentiating how their microstructures impact osteogenesis, in particular, for Ultrafine Grained (UFG) titanium. Then we identify key gaps in the research to date, pointing out areas which merit additional focus within the scientific community. For example, we highlight the potential of next-generation DNA sequencing techniques (NGS) to reveal gene and non-coding RNA (ncRNA) expression changes induced by nanostructured metals. While our understanding of bio-nano interactions is in its infancy, nanostructured metals are already being marketed or developed for medical devices such as dental implants, spinal devices, and coronary stents. Our ability to characterize and optimize the biological response of cells to SPD metals will have synergistic effects on advances in materials, biological, and medical science.

Virus purification and enrichment by hydroxyapatite chromatography on a chip

The spread of infectious diseases has become a global health concern. In order to diagnose infectious diseases quickly and accurately, next-generation DNA sequencing techniques for genetic analysis of infectious viruses have been developed rapidly. However, it takes a very long time to pretreat clinical samples for genetic analysis using next-generation sequencers. We have therefore developed a microfluidic chromatography chip that can purify and enrich viruses in a sample using hydroxyapatite particles packed in a micro-column. We demonstrated the purification of virus from a mixture of virus and FBS protein, and enrichment of the virus using this novel microfluidic chip.

Advances in genetic diagnosis of neurological disorders

Neurogenetics has developed enormously in recent years, and the genetic basis of human disorders is being unravelled rapidly. Many neurological disorders are Mendelian disorders, caused by mutations in genes involved in normal function of the brain, spinal cord, peripheral nerves or muscles. Due to high costs and time-consuming procedures, genetic tests have normally been performed late in the diagnostic process, when clinical examination and other tests have indicated a specific gene as the likely disease cause. Many neurological phenotypes are genetically very heterogeneous, and testing of all possible disease genes has been impossible. As a result, many patients with genetic neurological disorders have remained without a specific diagnosis, even when the disease is caused by mutations in known disease genes. Recent technological advances, in particular next-generation DNA sequencing techniques, have resulted in rapid identification of genes involved in Mendelian disorders and provided new possibilities for diagnostic genetic testing. The development of methods for coupling targeted capture and massively parallel DNA sequencing has made it possible to examine a large number of genes in a single reaction. Diagnostic genetic testing can today be performed by the use of gene panels and exome sequencing. This allows a more precise diagnosis of many neurological disorders, and genetic testing should now be considered earlier in the diagnostic procedure.

Regulation of gene expression by 1,25-dihydroxyvitamin D3 in bone cells: exploiting new approaches and defining new mechanisms

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) serve both to orchestrate calcium and phosphorus homeostasis in higher vertebrates and to regulate a diverse set of cellular functions unrelated to control of mineral metabolism. With regard to bone, mesenchymal lineage cells, including both early and late osteoblasts as well as osteocytes represent classic targets of the vitamin D hormone. Accordingly, much of the early information regarding our current understanding of the mechanism of action of 1,25(OH)2D3, of which gene regulation is central, derives from a broad array of studies in these cell types. Indeed, a gene that provided both the earliest and perhaps the most extensive information regarding this and additional mechanisms was that of osteoblast-specific osteocalcin. Subsequent work has provided much additional detail as to how 1,25(OH)2D3, through the vitamin D receptor (VDR), mediates the modulation of many bone cell genes. In recent years, however, a series of technical advances involving the coupling of chromatin immunoprecipitation (ChIP) to unbiased methodologies that involve next-generation DNA sequencing techniques (ChIP-seq) have opened new avenues in the study of gene regulation. In this review, we summarize early work and then focus on more recent studies that have used ChIP-seq analysis and other approaches to provide insight into not only the regulation of specific genes such as the VDR, TNFSF11 (RANKL), LRP5, CBS and CYP24a1, but overarching genome-wide principles of gene regulation as well. The results of these studies highlight the value of these new approaches and the increased insight that can be gained.

From genomics to functional markers in the era of next-generation sequencing

The availability of complete genome sequences, along with other genomic resources for Arabidopsis, rice, pigeon pea, soybean and other crops, has revolutionized our understanding of the genetic make-up of plants. Next-generation DNA sequencing (NGS) has facilitated single nucleotide polymorphism discovery in plants. Functionally-characterized sequences can be identified and functional markers (FMs) for important traits can be developed at an ever-increasing ease. FMs are derived from sequence polymorphisms found in allelic variants of a functional gene. Linkage disequilibrium-based association mapping and homologous recombinants have been developed for identification of "perfect" markers for their use in crop improvement practices. Compared with many other molecular markers, FMs derived from the functionally characterized sequence genes using NGS techniques and their use provide opportunities to develop high-yielding plant genotypes resistant to various stresses at a fast pace.

Imaging Epigenetics in Alzheimer’s Disease

Sporadic Alzheimer's disease (AD) is a prevalent, complex and chronically progressive brain disease. Its course is non-linear, dynamic, adaptive to maladaptive, and compensatory to decompensatory, affecting large-scale neural networks through a plethora of mechanistic and signaling pathway alterations that converge into regional and cell type-specific neurodegeneration and, finally, into clinically overt cognitive and behavioral decline. This decline includes reductions in the activities of daily living, quality of life, independence, and life expectancy. Evolving lines of research suggest that epigenetic mechanisms may play a crucial role during AD development and progression. Epigenetics designates molecular mechanisms that alter gene expression without modifications of the genetic code. This topic includes modifications on DNA and histone proteins, the primary elements of chromatin structure. Accumulating evidence has revealed the relevant processes that mediate epigenetic modifications and has begun to elucidate how these processes are apparently dysregulated in AD. This evidence has led to the clarification of the roles of specific classes of therapeutic compounds that affect epigenetic pathways and characteristics of the epigenome. This insight is accompanied by the development of new methods for studying the global patterns of DNA methylation and chromatin alterations. In particular, high-throughput sequencing approaches, such as next-generation DNA sequencing techniques, are beginning to drive the field into the next stage of development. In parallel, genetic imaging is beginning to answer additional questions through its ability to uncover genetic variants, with or without genome-wide significance, that are related to brain structure, function and metabolism, which impact disease risk and fundamental network-based cognitive processes. Neuroimaging measures can further be used to define AD systems and endophenotypes. The integration of genetic neuroimaging methods with epigenetic markers in humans appears promising. This evolving development may lead to a new research discipline - imaging epigenetics - that will provide deeper insight into the causative pathogenetic and pathophysiological pathways through which genes and environment interrelate during life and impact human brain development, physiology, aging and disease. This knowledge may open doors for the development of novel biomarkers and preventive and disease-modifying treatments.

Population Abundance of Potentially Pathogenic Organisms in Intestinal Microbiome of Jungle Crow (Corvus macrorhynchos) Shown with 16S rRNA Gene-Based Microbial Community Analysis

Jungle Crows (Corvus macrorhynchos) prefer human habitats because of their versatility in feeding accompanied with human food consumption. Therefore, it is important from a public health viewpoint to characterize their intestinal microbiota. However, no studies have been involved in molecular characterization of the microbiota based on huge and reliable number of data acquisition. In this study, 16S rRNA gene-based microbial community analysis coupled with the next-generation DNA sequencing techniques was applied to the taxonomic classification of intestinal microbiome for three jungle crows. Clustering of the reads into 130 operational taxonomic units showed that at least 70% of analyzed sequences for each crow were highly homologous to Eimeria sp., which belongs to the protozoan phylum Apicomplexa. The microbiotas of three crows also contained potentially pathogenic bacteria with significant percentages, such as the genera Campylobacter and Brachyspira. Thus, the profiling of a large number of 16S rRNA gene sequences in crow intestinal microbiomes revealed the high-frequency existence or vestige of potentially pathogenic microorganisms.

Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target amplification technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.

Developing genomic resources in two Linum species via 454 pyrosequencing and genomic reduction

Recent advances in next-generation DNA sequencing (NGS) have enhanced the development of genomic resources such as contigs or single-nucleotide polymorphisms (SNPs) for evolutionary studies of a nonmodel species with a complex and unsequenced genome. This study presents an application of a NGS technique in combination with genomic reduction and advanced bioinformatics tools to identify contigs and SNPs from multiple samples of two Linum species. A full Roche 454 GS FLX run of 16 diverse Linum samples representing cultivated flax (Linum usitatissimum L.) and its wild progenitor (Linum bienne Mill.) generated approximately 1.6 million sequence reads with a total length of 498 Mbp. Application of the computational pipeline de novo identification of alleles identified 713 contigs and 1067 SNPs. A blast search revealed alignments of all 713 contigs with 491 existing Linum scaffolds and gene annotations associated with 512 contigs. Sanger sequencing confirmed 95% of 79 selected contigs and 94% of 272 SNPs and identified 211 new SNPs and 19 new indels. The scored 454 SNP data were highly imbalanced for assayed samples. These findings not only are useful for evolutionary studies of Linum species but also help to illustrate the utility of NGS technologies in SNP discovery for nonmodel organisms.

At least it won’t hurt: the personal risks of antibiotic exposure

This review presents recent evidence regarding the adverse effects of antibiotic therapy mediated by collateral damage to commensal flora. Two major drivers have characterized recent research in this field: new perspectives into human microbiota afforded by next-generation DNA sequencing techniques and ongoing attention to antimicrobial resistance. New molecular techniques have illustrated that antibiotic therapy can disturb human microbiota, and that these changes are associated with infection. Concurrently, epidemiologic studies using patient-level data offer new insights into the role of antibiotics in the emergence, selection and spread of antimicrobial resistance, and Clostridium difficile infection (CDI).

An Integrated Genomic Approach to the Assessment and Treatment of Acute Myeloid Leukemia

Traditionally, new scientific advances have been applied quickly to the leukemias based on the ease with which relatively pure samples of malignant cells can be obtained. Currently, our arsenal of approaches used to characterize an individual's acute myeloid leukemia (AML) combines hematopathologic evaluation, flow cytometry, cytogenetic analysis, and molecular studies focused on a few key genes. The advent of high-throughput methods capable of full-genome evaluation presents new options for a revolutionary change in the way we diagnose, characterize, and treat AML. Next-generation DNA sequencing techniques allow full sequencing of a cancer genome or transcriptome, with the hope that this will be affordable for routine clinical care within the decade. Microarray-based testing will define gene and miRNA expression, DNA methylation patterns, chromosomal imbalances, and predisposition to disease and chemosensitivity. The vision for the future entails an integrated and automated approach to these analyses, bringing the possibility of formulating an individualized treatment plan within days of a patient's initial presentation. With these expectations comes the hope that such an approach will lead to decreased toxicities and prolonged survival for patients.