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September 22, 2019

Research benefits of storing genitourinary samples: 16S rRNA sequencing to evaluate vaginal bacterial communities.

Using well-characterised, but old and carefully frozen genital tract research samples may be a cost-effective way of performing metagenomic studies, but risks loss of low abundance (but relevant) bacterial species DNA. Moi et al.1 used 16S rRNA and UreDNA sequencing to detect ureaplasmas in frozen urine samples collected from 362 men with NGU in 2010–2011. They found that urethral inflammatory responses to ureaplasmas were less severe than to Chlamydia trachomatis and Mycoplasma genitalium.

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September 22, 2019

Meeting report: processing, translation, decay – three ways to keep RNA sizzling.

This meeting report highlights key trends that emerged from a conference entitled Post-Transcriptional Gene Regulation in Plants, which was held 14-15 July 2016, as a satellite meeting of the annual meeting of the American Society of Plant Biologists in Austin, Texas. The molecular biology of RNA is emerging as an integral part of the framework for plants’ responses to environmental challenges such as drought and heat, hypoxia, nutrient deprivation, light and pathogens. Moreover, the conference illustrated how a multitude of customized and pioneering omics-related technologies are being applied, more and more often in combination, to describe and dissect the complexities of gene expression at the post-transcriptional level.© 2016 John Wiley & Sons Ltd.

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September 22, 2019

Indoleacrylic acid produced by commensal Peptostreptococcus species suppresses inflammation.

Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

Species groups distributed across elevational gradients reveal convergent and continuous genetic adaptation to high elevations.

Although many cases of genetic adaptations to high elevations have been reported, the processes driving these modifications and the pace of their evolution remain unclear. Many high-elevation adaptations (HEAs) are thought to have arisen in situ as populations rose with growing mountains. In contrast, most high-elevation lineages of the Qinghai-Tibetan Plateau appear to have colonized from low-elevation areas. These lineages provide an opportunity for studying recent HEAs and comparing them with ancestral low-elevation alternatives. Herein, we compare four frogs (three species of Nanorana and a close lowland relative) and four lizards (Phrynocephalus) that inhabit a range of elevations on or along the slopes of the Qinghai-Tibetan Plateau. The sequential cladogenesis of these species across an elevational gradient allows us to examine the gradual accumulation of HEA at increasing elevations. Many adaptations to high elevations appear to arise gradually and evolve continuously with increasing elevational distributions. Numerous related functions, especially DNA repair and energy metabolism pathways, exhibit rapid change and continuous positive selection with increasing elevations. Although the two studied genera are distantly related, they exhibit numerous convergent evolutionary changes, especially at the functional level. This functional convergence appears to be more extensive than convergence at the individual gene level, although we found 32 homologous genes undergoing positive selection for change in both high-elevation groups. We argue that species groups distributed along a broad elevational gradient provide a more powerful system for testing adaptations to high-elevation environments compared with studies that compare only pairs of high-elevation versus low-elevation species.

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September 22, 2019

Avian transcriptomics: opportunities and challenges

Recent developments in next-generation sequencing technologies have greatly facilitated the study of whole transcriptomes in model and non-model species. Studying the transcriptome and how it changes across a variety of biological conditions has had major implications for our understanding of how the genome is regulated in different contexts, and how to interpret adaptations and the phenotype of an organism. The aim of this review is to highlight the potential of these new technologies for the study of avian transcriptomics, and to summarise how transcriptomics has been applied in ornithology. A total of 81 peer-reviewed scientific articles that used transcriptomics to answer questions within a broad range of study areas in birds are used as examples throughout the review. We further provide a quick guide to highlight the most important points which need to be take into account when planning a transcriptomic study in birds, and discuss how researchers with little background in molecular biology can avoid potential pitfalls. Suggestions for further reading are supplied throughout. We also discuss possible future developments in the technology platforms used for ribonucleic acid sequencing. By summarising how these novel technologies can be used to answer questions that have long been asked by ornithologists, we hope to bridge the gap between traditional ornithology and genomics, and to stimulate more interdisciplinary research.

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September 22, 2019

Single-molecule long-read 16S sequencing to characterize the lung microbiome from mechanically ventilated patients with suspected pneumonia.

In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

Full-length transcriptome sequences of ephemeral plant Arabidopsis pumila provides insight into gene expression dynamics during continuous salt stress.

Arabidopsis pumila is native to the desert region of northwest China and it is extraordinarily well adapted to the local semi-desert saline soil, thus providing a candidate plant system for environmental adaptation and salt-tolerance gene mining. However, understanding of the salt-adaptation mechanism of this species is limited because of genomic sequences scarcity. In the present study, the transcriptome profiles of A. pumila leaf tissues treated with 250 mM NaCl for 0, 0.5, 3, 6, 12, 24 and 48 h were analyzed using a combination of second-generation sequencing (SGS) and third-generation single-molecule real-time (SMRT) sequencing.Correction of SMRT long reads by SGS short reads resulted in 59,328 transcripts. We found 8075 differentially expressed genes (DEGs) between salt-stressed tissues and controls, of which 483 were transcription factors and 1157 were transport proteins. Most DEGs were activated within 6 h of salt stress and their expression stabilized after 48 h; the number of DEGs was greatest within 12 h of salt stress. Gene annotation and functional analyses revealed that expression of genes associated with the osmotic and ionic phases rapidly and coordinately changed during the continuous salt stress in this species, and salt stress-related categories were highly enriched among these DEGs, including oxidation-reduction, transmembrane transport, transcription factor activity and ion channel activity. Orphan, MYB, HB, bHLH, C3H, PHD, bZIP, ARF and NAC TFs were most enriched in DEGs; ABCB1, CLC-A, CPK30, KEA2, KUP9, NHX1, SOS1, VHA-A and VP1 TPs were extensively up-regulated in salt-stressed samples, suggesting that they play important roles in slat tolerance. Importantly, further experimental studies identified a mitogen-activated protein kinase (MAPK) gene MAPKKK18 as continuously up-regulated throughout salt stress, suggesting its crucial role in salt tolerance. The expression patterns of the salt-responsive 24 genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-Seq.The full-length transcripts generated in this study provide a more accurate depiction of gene transcription of A. pumila. We identified potential genes involved in salt tolerance of A. pumila. These data present a genetic resource and facilitate better understanding of salt-adaptation mechanism for ephemeral plants.

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September 22, 2019

Long-term changes of bacterial and viral compositions in the intestine of a recovered Clostridium difficile patient after fecal microbiota transplantation

Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infections (RCDIs). However, long-term effects on the patients’ gut microbiota and the role of viruses remain to be elucidated. Here, we characterized bacterial and viral microbiota in the feces of a cured RCDI patient at various time points until 4.5 yr post-FMT compared with the stool donor. Feces were subjected to DNA sequencing to characterize bacteria and double-stranded DNA (dsDNA) viruses including phages. The patient’s microbial communities varied over time and showed little overall similarity to the donor until 7 mo post-FMT, indicating ongoing gut microbiota adaption in this time period. After 4.5 yr, the patient’s bacteria attained donor-like compositions at phylum, class, and order levels with similar bacterial diversity. Differences in the bacterial communities between donor and patient after 4.5 yr were seen at lower taxonomic levels. C. difficile remained undetectable throughout the entire timespan. This demonstrated sustainable donor feces engraftment and verified long-term therapeutic success of FMT on the molecular level. Full engraftment apparently required longer than previously acknowledged, suggesting the implementation of year-long patient follow-up periods into clinical practice. The identified dsDNA viruses were mainly Caudovirales phages. Unexpectedly, sequences related to giant algae–infecting Chlorella viruses were also detected. Our findings indicate that intestinal viruses may be implicated in the establishment of gut microbiota. Therefore, virome analyses should be included in gut microbiota studies to determine the roles of phages and other viruses—such as Chlorella viruses—in human health and disease, particularly during RCDI.

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September 22, 2019

Genomic insights into the acid adaptation of novel methanotrophs enriched from acidic forest soils.

Soil acidification is accelerated by anthropogenic and agricultural activities, which could significantly affect global methane cycles. However, detailed knowledge of the genomic properties of methanotrophs adapted to acidic soils remains scarce. Using metagenomic approaches, we analyzed methane-utilizing communities enriched from acidic forest soils with pH 3 and 4, and recovered near-complete genomes of proteobacterial methanotrophs. Novel methanotroph genomes designated KS32 and KS41, belonging to two representative clades of methanotrophs (Methylocystis of Alphaproteobacteria and Methylobacter of Gammaproteobacteria), were dominant. Comparative genomic analysis revealed diverse systems of membrane transporters for ensuring pH homeostasis and defense against toxic chemicals. Various potassium transporter systems, sodium/proton antiporters, and two copies of proton-translocating F1F0-type ATP synthase genes were identified, which might participate in the key pH homeostasis mechanisms in KS32. In addition, the V-type ATP synthase and urea assimilation genes might be used for pH homeostasis in KS41. Genes involved in the modification of membranes by incorporation of cyclopropane fatty acids and hopanoid lipids might be used for reducing proton influx into cells. The two methanotroph genomes possess genes for elaborate heavy metal efflux pumping systems, possibly owing to increased heavy metal toxicity in acidic conditions. Phylogenies of key genes involved in acid adaptation, methane oxidation, and antiviral defense in KS41 were incongruent with that of 16S rRNA. Thus, the detailed analysis of the genome sequences provides new insights into the ecology of methanotrophs responding to soil acidification.

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September 22, 2019

Meeting report: 31st International Mammalian Genome Conference, Mammalian Genetics and Genomics: From Molecular Mechanisms to Translational Applications.

High on the Heidelberg hills, inside the Advanced Training Centre of the European Molecular Biology Laboratory (EMBL) campus with its unique double-helix staircase, scientists gathered for the EMBL conference “Mammalian Genetics and Genomics: From Molecular Mechanisms to Translational Applications,” organized in cooperation with the International Mammalian Genome Society (IMGS) and the Mouse Molecular Genetics (MMG) group. The conference attracted 205 participants from 30 countries, representing 6 of the 7 continents-all except Antarctica. It was a richly diverse group of geneticists, clinicians, and bioinformaticians, with presentations by established and junior investigators, including many trainees. From the 24th-27th of October 2017, they shared exciting advances in mammalian genetics and genomics research, from the introduction of cutting-edge technologies to descriptions of translational studies involving highly relevant models of human disease.

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September 22, 2019

A community-based culture collection for targeting novel plant growth-promoting bacteria from the sugarcane microbiome.

The soil-plant ecosystem harbors an immense microbial diversity that challenges investigative approaches to study traits underlying plant-microbe association. Studies solely based on culture-dependent techniques have overlooked most microbial diversity. Here we describe the concomitant use of culture-dependent and -independent techniques to target plant-beneficial microbial groups from the sugarcane microbiome. The community-based culture collection (CBC) approach was used to access microbes from roots and stalks. The CBC recovered 399 unique bacteria representing 15.9% of the rhizosphere core microbiome and 61.6-65.3% of the endophytic core microbiomes of stalks. By cross-referencing the CBC (culture-dependent) with the sugarcane microbiome profile (culture-independent), we designed a synthetic community comprised of naturally occurring highly abundant bacterial groups from roots and stalks, most of which has been poorly explored so far. We then used maize as a model to probe the abundance-based synthetic inoculant. We show that when inoculated in maize plants, members of the synthetic community efficiently colonize plant organs, displace the natural microbiota and dominate at 53.9% of the rhizosphere microbial abundance. As a result, inoculated plants increased biomass by 3.4-fold as compared to uninoculated plants. The results demonstrate that abundance-based synthetic inoculants can be successfully applied to recover beneficial plant microbes from plant microbiota.

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September 22, 2019

Full-length transcriptome of Misgurnus anguillicaudatus provides insights into evolution of genus Misgurnus.

Reconstruction and annotation of transcripts, particularly for a species without reference genome, plays a critical role in gene discovery, investigation of genomic signatures, and genome annotation in the pre-genomic era. This study generated 33,330 full-length transcripts of diploid M. anguillicaudatus using PacBio SMRT Sequencing. A total of 6,918 gene families were identified with two or more isoforms, and 26,683 complete ORFs with an average length of 1,497?bp were detected. Totally, 1,208 high-confidence lncRNAs were identified, and most of these appeared to be precursor transcripts of miRNAs or snoRNAs. Phylogenetic tree of the Misgurnus species was inferred based on the 1,905 single copy orthologous genes. The tetraploid and diploid M. anguillicaudatus grouped into a clade, and M. bipartitus showed a closer relationship with the M. anguillicaudatus. The overall evolutionary rates of tetraploid M. anguillicaudatus were significantly higher than those of other Misgurnus species. Meanwhile, 28 positively selected genes were identified in M. anguillicaudatus clade. These positively selected genes may play critical roles in the adaptation to various habitat environments for M. anguillicaudatus. This study could facilitate further exploration of the genomic signatures of M. anguillicaudatus and provide potential insights into unveiling the evolutionary history of tetraploid loach.

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September 22, 2019

Bacteroides dorei dominates gut microbiome prior to autoimmunity in Finnish children at high risk for type 1 diabetes.

The incidence of the autoimmune disease, type 1 diabetes (T1D), has increased dramatically over the last half century in many developed countries and is particularly high in Finland and other Nordic countries. Along with genetic predisposition, environmental factors are thought to play a critical role in this increase. As with other autoimmune diseases, the gut microbiome is thought to play a potential role in controlling progression to T1D in children with high genetic risk, but we know little about how the gut microbiome develops in children with high genetic risk for T1D. In this study, the early development of the gut microbiomes of 76 children at high genetic risk for T1D was determined using high-throughput 16S rRNA gene sequencing. Stool samples from children born in the same hospital in Turku, Finland were collected at monthly intervals beginning at 4-6 months after birth until 2.2 years of age. Of those 76 children, 29 seroconverted to T1D-related autoimmunity (cases) including 22 who later developed T1D, the remaining 47 subjects remained healthy (controls). While several significant compositional differences in low abundant species prior to seroconversion were found, one highly abundant group composed of two closely related species, Bacteroides dorei and Bacteroides vulgatus, was significantly higher in cases compared to controls prior to seroconversion. Metagenomic sequencing of samples high in the abundance of the B. dorei/vulgatus group before seroconversion, as well as longer 16S rRNA sequencing identified this group as Bacteroides dorei. The abundance of B. dorei peaked at 7.6 months in cases, over 8 months prior to the appearance of the first islet autoantibody, suggesting that early changes in the microbiome may be useful for predicting T1D autoimmunity in genetically susceptible infants. The cause of increased B. dorei abundance in cases is not known but its timing appears to coincide with the introduction of solid food.

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September 22, 2019

Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire.

Vertebrate immune systems generate diverse repertoires of antibodies capable of mediating response to a variety of antigens. Next generation sequencing methods provide unique approaches to a number of immuno-based research areas including antibody discovery and engineering, disease surveillance, and host immune response to vaccines. In particular, single-molecule circular consensus sequencing permits the sequencing of antibody repertoires at previously unattainable depths of coverage and accuracy. We approached the bovine immunoglobulin G (IgG) repertoire with the objective of characterizing diversity of expressed IgG transcripts. Here we present single-molecule real-time sequencing data of expressed IgG heavy-chain repertoires of four individual cattle. We describe the diversity observed within antigen binding regions and visualize this diversity using a network-based approach.We generated 49,945 high quality cDNA sequences, each spanning the entire IgG variable region from four Bos taurus calves. From these sequences we identified 49,521 antigen binding regions using the automated Paratome web server. Approximately 9% of all unique complementarity determining 2 (CDR2) sequences were of variable lengths. A bimodal distribution of unique CDR3 sequence lengths was observed, with common lengths of 5-6 and 21-25 amino acids. The average number of cysteine residues in CDR3s increased with CDR3 length and we observed that cysteine residues were centrally located in CDR3s. We identified 19 extremely long CDR3 sequences (up to 62 amino acids in length) within IgG transcripts. Network analyses revealed distinct patterns among the expressed IgG antigen binding repertoires of the examined individuals.We utilized circular consensus sequencing technology to provide baseline data of the expressed bovine IgG repertoire that can be used for future studies important to livestock research. Somatic mutation resulting in base insertions and deletions in CDR2 further diversifies the bovine antibody repertoire. In contrast to previous studies, our data indicate that unusually long CDR3 sequences are not unique to IgM antibodies in cattle. Centrally located cysteine residues in bovine CDR3s provide further evidence that disulfide bond formation is likely of structural importance. We hypothesize that network or cluster-based analyses of expressed antibody repertoires from controlled challenge experiments will help identify novel natural antigen binding solutions to specific pathogens of interest.

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September 22, 2019

Fungal ITS1 deep-sequencing strategies to reconstruct the composition of a 26-species community and evaluation of the gut mycobiota of healthy Japanese individuals.

The study of mycobiota remains relatively unexplored due to the lack of sufficient available reference strains and databases compared to those of bacterial microbiome studies. Deep sequencing of Internal Transcribed Spacer (ITS) regions is the de facto standard for fungal diversity analysis. However, results are often biased because of the wide variety of sequence lengths in the ITS regions and the complexity of high-throughput sequencing (HTS) technologies. In this study, a curated ITS database, ntF-ITS1, was constructed. This database can be utilized for the taxonomic assignment of fungal community members. We evaluated the efficacy of strategies for mycobiome analysis by using this database and characterizing a mock fungal community consisting of 26 species representing 15 genera using ITS1 sequencing with three HTS platforms: Illumina MiSeq (MiSeq), Ion Torrent Personal Genome Machine (IonPGM), and Pacific Biosciences (PacBio). Our evaluation demonstrated that PacBio’s circular consensus sequencing with greater than 8 full-passes most accurately reconstructed the composition of the mock community. Using this strategy for deep-sequencing analysis of the gut mycobiota in healthy Japanese individuals revealed two major mycobiota types: a single-species type composed of Candida albicans or Saccharomyces cerevisiae and a multi-species type. In this study, we proposed the best possible processing strategies for the three sequencing platforms, of which, the PacBio platform allowed for the most accurate estimation of the fungal community. The database and methodology described here provide critical tools for the emerging field of mycobiome studies.

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