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September 22, 2019

Identification of microbial profile of Koji using Single Molecule, Real-Time Sequencing technology.

Koji is a kind of Japanese traditional fermented starter that has been used for centuries. Many fermented foods are made from koji, such as sake, miso, and soy sauce. This study used the single molecule real-time sequencing technology (SMRT) to investigate the bacterial and fungal microbiota of 3 Japanese koji samples. After SMRT analysis, a total of 39121 high-quality sequences were generated, including 14354 bacterial and 24767 fungal sequence reads. The high-quality gene sequences were assigned to 5 bacterial and 2 fungal plyla, dominated by Proteobacteria and Ascomycota, respectively. At the genus level, Ochrobactrum and Wickerhamomyces were the most abundant bacterial and fungal genera, respectively. The predominant bacterial and fungal species were Ochrobactrum lupini and Wickerhamomyces anomalus, respectively. Our study profiled the microbiota composition of 3 Japanese koji samples to the species level precision. The results may be useful for further development of traditional fermented products, especially optimization of koji preparation. Meanwhile, this study has demonstrated that SMRT is a robust tool for analyzing the microbial composition in food samples.© 2017 Institute of Food Technologists®.

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September 22, 2019

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.

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September 22, 2019

Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.

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September 22, 2019

Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

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September 22, 2019

Initial colonization, community assembly and ecosystem function: fungal colonist traits and litter biochemistry mediate decay rate.

Priority effects are an important ecological force shaping biotic communities and ecosystem processes, in which the establishment of early colonists alters the colonization success of later-arriving organisms via competitive exclusion and habitat modification. However, we do not understand which biotic and abiotic conditions lead to strong priority effects and lasting historical contingencies. Using saprotrophic fungi in a model leaf decomposition system, we investigated whether compositional and functional consequences of initial colonization were dependent on initial colonizer traits, resource availability or a combination thereof. To test these ideas, we factorially manipulated leaf litter biochemistry and initial fungal colonist identity, quantifying subsequent community composition, using neutral genetic markers, and community functional characteristics, including enzyme potential and leaf decay rates. During the first 3 months, initial colonist respiration rate and physiological capacity to degrade plant detritus were significant determinants of fungal community composition and leaf decay, indicating that rapid growth and lignolytic potential of early colonists contributed to altered trajectories of community assembly. Further, initial colonization on oak leaves generated increasingly divergent trajectories of fungal community composition and enzyme potential, indicating stronger initial colonizer effects on energy-poor substrates. Together, these observations provide evidence that initial colonization effects, and subsequent consequences on litter decay, are dependent upon substrate biochemistry and physiological traits within a regional species pool. Because microbial decay of plant detritus is important to global C storage, our results demonstrate that understanding the mechanisms by which initial conditions alter priority effects during community assembly may be key to understanding the drivers of ecosystem-level processes. © 2015 John Wiley & Sons Ltd.

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September 22, 2019

Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin.

The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3?m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.

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September 22, 2019

Scale-up of sediment microbial fuel cells.

Sediment microbial fuel cells (SMFCs) are used as renewable power sources to operate remote sensors. However, increasing the electrode surface area results in decreased power density, which demonstrates that SMFCs do not scale up with size. As an alternative to the physical scale-up of SMFCs, we proposed that it is possible to scale up power by using smaller-sized individually operated SMFCs connected to a power management system that electrically isolates the anodes and cathodes. To demonstrate our electronic scale-up approach, we operated one 0.36-m2 SMFC (called a single-equivalent SMFC) and four independent SMFCs of 0.09 m2 each (called scaled-up SMFCs) and managed the power using an innovative custom-developed power management system. We found that the single-equivalent SMFC and the scaled-up SMFCs produced similar power for the first 155 days. However, in the long term (>155 days) our scaled-up SMFCs generated significantly more power than the single-equivalent SMFC (2.33 mW vs. 0.64 mW). Microbial community analysis of the single-equivalent SMFC and the scaled-up SMFCs showed very similar results, demonstrating that the difference in operation mode had no significant effect on the microbial community. When we compared scaled-up SMFCs with parallel SMFCs, we found that the scaled-up SMFCs generated more power. Our novel approach demonstrates that SMFCs can be scaled up electronically.

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September 22, 2019

Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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September 22, 2019

The effects of probiotics administration on the milk production, milk components and fecal bacteria microbiota of dairy cows

Probiotics administration can improve host health. This study aims to determine the effects of probiotics (Lactobacillus casei Zhang and Lactobacillus plantarum P-8) administration on milk production, milk functional components, milk composition, and fecal microbiota of dairy cows. Variations in the fecal bacteria microbiota between treatments were assessed based on 16S rRNA profiles determined by PacBio single molecule real-time sequencing technology. The probiotics supplementation significantly increased the milk production and the contents of milk immunoglobulin G (IgG), lactoferrin (LTF), lysozyme (LYS) and lactoperoxidase (LP), while the somatic cell counts (SCC) significantly decreased (P < 0.01). However, no significant difference was found in the milk fat, protein and lactose contents (P > 0.05). Although the probiotics supplementation did not change the fecal bacteria richness and diversity, significantly more rumen fermentative bacteria (Bacteroides, Roseburia, Ruminococcus, Clostridium, Coprococcus and Dorea) and beneficial bacteria (Faecalibacterium prausnitzii) were found in the probiotics treatment group. Meanwhile, some opportunistic pathogens e.g. Bacillus cereus, Cronobacter sakazakii and Alkaliphilus oremlandii, were suppressed. Additionally, we found some correlations between the milk production, milk components and fecal bacteria. To sum up, our study demonstrated the beneficial effects of probiotics application in improving the quality and quantity of cow milk production.

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September 22, 2019

Complex effects of mammalian grazing on extramatrical mycelial biomass in the Scandes forest-tundra ecotone.

Mycorrhizal associations are widespread in high-latitude ecosystems and are potentially of great importance for global carbon dynamics. Although large herbivores play a key part in shaping subarctic plant communities, their impact on mycorrhizal dynamics is largely unknown. We measured extramatrical mycelial (EMM) biomass during one growing season in 16-year-old herbivore exclosures and unenclosed control plots (ambient), at three mountain birch forests and two shrub heath sites, in the Scandes forest-tundra ecotone. We also used high-throughput amplicon sequencing for taxonomic identification to investigate differences in fungal species composition. At the birch forest sites, EMM biomass was significantly higher in exclosures (1.36 ± 0.43 g C/m2) than in ambient conditions (0.66 ± 0.17 g C/m2) and was positively influenced by soil thawing degree-days. At the shrub heath sites, there was no significant effect on EMM biomass (exclosures: 0.72 ± 0.09 g C/m2; ambient plots: 1.43 ± 0.94). However, EMM biomass was negatively related toBetula nanaabundance, which was greater in exclosures, suggesting that grazing affected EMM biomass positively. We found no significant treatment effects on fungal diversity but the most abundant ectomycorrhizal lineage/cortinarius, showed a near-significant positive effect of herbivore exclusion (p = .08), indicating that herbivory also affects fungal community composition. These results suggest that herbivory can influence fungal biomass in highly context-dependent ways in subarctic ecosystems. Considering the importance of root-associated fungi for ecosystem carbon balance, these findings could have far-reaching implications.

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September 22, 2019

Transcriptome-referenced association study of clove shape traits in garlic.

Genome-wide association studies are a powerful approach for identifying genes related to complex traits in organisms, but are limited by the requirement for a reference genome sequence of the species under study. To circumvent this problem, we propose a transcriptome-referenced association study (TRAS) that utilizes a transcriptome generated by single-molecule long-read sequencing as a reference sequence to score population variation at both transcript sequence and expression levels. Candidate transcripts are identified when both scores are associated with a trait and their potential interactions are ascertained by expression quantitative trait loci analysis. Applying this method to characterize garlic clove shape traits in 102 landraces, we identified 22 candidate transcripts, most of which showed extensive interactions. Eight transcripts were long non-coding RNAs (lncRNAs), and the others were proteins involved mainly in carbohydrate metabolism, protein degradation, etc. TRAS, as an efficient tool for association study independent of a reference genome, extends the applicability of association studies to a broad range of species.

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September 22, 2019

High-resolution expression map of the Arabidopsis root reveals alternative splicing and lincRNA regulation.

The extent to which alternative splicing and long intergenic noncoding RNAs (lincRNAs) contribute to the specialized functions of cells within an organ is poorly understood. We generated a comprehensive dataset of gene expression from individual cell types of the Arabidopsis root. Comparisons across cell types revealed that alternative splicing tends to remove parts of coding regions from a longer, major isoform, providing evidence for a progressive mechanism of splicing. Cell-type-specific intron retention suggested a possible origin for this common form of alternative splicing. Coordinated alternative splicing across developmental stages pointed to a role in regulating differentiation. Consistent with this hypothesis, distinct isoforms of a transcription factor were shown to control developmental transitions. lincRNAs were generally lowly expressed at the level of individual cell types, but co-expression clusters provided clues as to their function. Our results highlight insights gained from analysis of expression at the level of individual cell types. Copyright © 2016 Elsevier Inc. All rights reserved.

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September 22, 2019

Anthropogenic N deposition alters the composition of expressed class II fungal peroxidases.

Here, we present evidence that ca. 20 years of experimental N deposition altered the composition of lignin-decaying class II peroxidases expressed by forest floor fungi, a response which has occurred concurrently with reductions in plant litter decomposition and a rapid accumulation of soil organic matter. This finding suggests that anthropogenic N deposition has induced changes in the biological mediation of lignin decay, the rate limiting step in plant litter decomposition. Thus, an altered composition of transcripts for a critical gene that is associated with terrestrial C cycling may explain the increased soil C storage under long-term increases in anthropogenic N deposition.IMPORTANCE Fungal class II peroxidases are enzymes that mediate the rate-limiting step in the decomposition of plant material, which involves the oxidation of lignin and other polyphenols. In field experiments, anthropogenic N deposition has increased soil C storage in forests, a result which could potentially arise from anthropogenic N-induced changes in the composition of class II peroxidases expressed by the fungal community. In this study, we have gained unique insight into how anthropogenic N deposition, a widespread agent of global change, affects the expression of a functional gene encoding an enzyme that plays a critical role in a biologically mediated ecosystem process. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Single-molecule long-read transcriptome dataset of halophyte Halogeton glomeratus.

Soil salinization has become a major challenge for sustainable development of global agriculture. As a result, cultivation of salt-tolerant crop varieties has become a focus of plant breeding. However, development of effective breeding strategies would be significantly enhanced by improving our understanding of salt tolerance mechanisms in plants and identifying genes required for adaptation.

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September 22, 2019

Atmospheric N deposition increases bacterial laccase-like multicopper oxidases: implications for organic matter decay.

Anthropogenic release of biologically available nitrogen (N) has increased dramatically over the last 150 years, which can alter the processes controlling carbon (C) storage in terrestrial ecosystems. In a northern hardwood forest ecosystem located in Michigan in the United States, nearly 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This change occurred concomitantly with compositional changes in Basidiomycete fungi and in Actinobacteria, as well as the downregulation of fungal lignocelluloytic genes. Recently, laccase-like multicopper oxidases (LMCOs) have been discovered among bacteria which can oxidize ß-O-4 linkages in phenolic compounds (e.g., lignin and humic compounds), resulting in the production of dissolved organic carbon (DOC). Here, we examined how nearly 2 decades of experimental N deposition has affected the abundance and composition of saprotrophic bacteria possessing LMCO genes. In our experiment, LMCO genes were more abundant in the forest floor under experimental N deposition whereas the abundances of bacteria and fungi were unchanged. Experimental N deposition also led to less-diverse, significantly altered bacterial and LMCO gene assemblages, with taxa implicated in organic matter decay (i.e., Actinobacteria, Proteobacteria) accounting for the majority of compositional changes. These results suggest that experimental N deposition favors bacteria in the forest floor that harbor the LMCO gene and represents a plausible mechanism by which anthropogenic N deposition has reduced decomposition, increased soil C storage, and accelerated phenolic DOC production in our field experiment. Our observations suggest that future rates of atmospheric N deposition could fundamentally alter the physiological potential of soil microbial communities. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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