Menu

Auto Tag: Microbiology

September 22, 2019  |  

HIV-1 infection of primary CD4(+) T cells regulates the expression of specific HERV-K (HML-2) elements.

Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathologic states, such as viral infections and certain cancers, coincide with ERV expression suggesting transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but the conserved nature of these elements has, until recently, rendered their expression profiling problematic.Here, we provide comprehensive HERV-K HML-2 expression profiles specific for productively HIV-1 infected primary human CD4(+) T cells. We combined enrichment of HIV-1 infected cells using a reporter virus expressing a surface reporter for gentle and efficient purification with long-read Single Molecule Real-Time sequencing. We show that three HML-2 proviruses, 6q25.1, 8q24.3, and 19q13.42 are up-regulated on average between 3- and 5-fold in HIV-1 infected CD4(+) T cells. One provirus, HML-2 12q24.33, in contrast, was repressed in the presence of active HIV replication.In conclusion, this report identifies the HERV-K HML-2 loci whose expression profiles differ upon HIV-1 infection in primary human CD4(+) T cells. These data will help pave the way for further studies on the influence of endogenous retroviruses on HIV-1 replication.Importance Endogenous retroviruses inhabit big portions of our genome. And although they are mainly inert some of the evolutionarily younger members maintain the ability to express both RNA as well as proteins. We have developed an approach using long-read SMRT sequencing that produces long reads, that provides us with ability to obtain detailed and accurate HERV-K HML-2 expression profiles. We have now applied this approach to study HERV-K expression in the presence and absence of productive HIV-1 infection of primary human CD4(+) T cells. In addition to using SMRT sequencing, our strategy also includes the magnetic selection of the infected cells so that levels of background expression due to uninfected cells are kept at a minimum. The results in this manuscript provide the blueprint for in-depth studies of the interactions of the authentic upregulated HERV-K HML-2 elements and HIV-1. Copyright © 2017 American Society for Microbiology.

Read more
September 22, 2019  |  

Global transcript structure resolution of high gene density genomes through multi-platform data integration.

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Read more
September 22, 2019  |  

Subaerial biofilms on granitic historic buildings: microbial diversity and development of phototrophic multi-species cultures.

Microbial communities of natural subaerial biofilms developed on granitic historic buildings of a World Heritage Site (Santiago de Compostela, NW Spain) were characterized and cultured in liquid BG11 medium. Environmental barcoding through next-generation sequencing (Pacific Biosciences) revealed that the biofilms were mainly composed of species of Chlorophyta (green algae) and Ascomycota (fungi) commonly associated with rock substrata. Richness and diversity were higher for the fungal than for the algal assemblages and fungi showed higher heterogeneity among samples. Cultures derived from natural biofilms showed the establishment of stable microbial communities mainly composed of Chlorophyta and Cyanobacteria. Although most taxa found in these cultures were not common in the original biofilms, they are likely common pioneer colonizers of building stone surfaces, including granite. Stable phototrophic multi-species cultures of known microbial diversity were thus obtained and their reliability to emulate natural colonization on granite should be confirmed in further experiments.

Read more
September 22, 2019  |  

Improved performance of the PacBio SMRT technology for 16S rDNA sequencing.

Improved sequencing accuracy was obtained with 16S amplicons from environmental samples and a known pure culture when upgraded Pacific Biosciences (PacBio) hardware and enzymes were used for the single molecule, real-time (SMRT) sequencing platform. The new PacBio RS II system with P4/C2 chemistry, when used with previously constructed libraries (Mosher et al., 2013) surpassed the accuracy of Roche/454 pyrosequencing platform. With accurate read lengths of >1400 base pairs, the PacBio system opens up the possibility of identifying microorganisms to the species level in environmental samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Read more
September 22, 2019  |  

A novel lactobacilli-based teat disinfectant for improving bacterial communities in the milks of cow teats with subclinical mastitis.

Teat disinfection pre- and post-milking is important for the overall health and hygiene of dairy cows. The objective of this study was to evaluate the efficacy of a novel probiotic lactobacilli-based teat disinfectant based on changes in somatic cell count (SCC) and profiling of the bacterial community. A total of 69 raw milk samples were obtained from eleven Holstein-Friesian dairy cows over 12 days of teat dipping in China. Single molecule, real-time sequencing technology (SMRT) was employed to profile changes in the bacterial community during the cleaning protocol and to compare the efficacy of probiotic lactic acid bacteria (LAB) and commercial teat disinfectants. The SCC gradually decreased following the cleaning protocol and the SCC of the LAB group was slightly lower than that of the commercial disinfectant (CD) group. Our SMRT sequencing results indicate that raw milk from both the LAB and CD groups contained diverse microbial populations that changed over the course of the cleaning protocol. The relative abundances of some species were significantly changed during the cleaning process, which may explain the observed bacterial community differences. Collectively, these results suggest that the LAB disinfectant could reduce mastitis-associated bacteria and improve the microbial environment of the cow teat. It could be used as an alternative to chemical pre- and post-milking teat disinfectants to maintain healthy teats and udders. In addition, the Pacific Biosciences SMRT sequencing with the full-length 16S ribosomal RNA gene was shown to be a powerful tool for monitoring changes in the bacterial population during the cleaning protocol.

Read more
September 22, 2019  |  

Rapid infectious disease identification by next-generation DNA sequencing.

Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

Read more
September 22, 2019  |  

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales. Two distinct nearly full-length Rhizobiales genomes were identified in leaf-pocket-enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2 -fixation. 15 N2 incorporation was active in ferns with N. azollae but not in ferns without. N2 O was not detectably released from surface-sterilized ferns with the Rhizobiales. N2 -fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2 O.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Read more
September 22, 2019  |  

Draft genome sequence of Sulfurospirillum sp. strain MES, reconstructed from the metagenome of a microbial electrosynthesis system.

A draft genome of Sulfurospirillum sp. strain MES was isolated through taxonomic binning of a metagenome sequenced from a microbial electrosynthesis system (MES) actively producing acetate and hydrogen. The genome contains the nosZDFLY genes, which are involved in nitrous oxide reduction, suggesting the potential role of this strain in denitrification. Copyright © 2015 Ross et al.

Read more
September 22, 2019  |  

Moving beyond microbiome-wide associations to causal microbe identification.

Microbiome-wide association studies have established that numerous diseases are associated with changes in the microbiota. These studies typically generate a long list of commensals implicated as biomarkers of disease, with no clear relevance to disease pathogenesis. If the field is to move beyond correlations and begin to address causation, an effective system is needed for refining this catalogue of differentially abundant microbes and to allow subsequent mechanistic studies. Here we demonstrate that triangulation of microbe-phenotype relationships is an effective method for reducing the noise inherent in microbiota studies and enabling identification of causal microbes. We found that gnotobiotic mice harbouring different microbial communities exhibited differential survival in a colitis model. Co-housing of these mice generated animals that had hybrid microbiotas and displayed intermediate susceptibility to colitis. Mapping of microbe-phenotype relationships in parental mouse strains and in mice with hybrid microbiotas identified the bacterial family Lachnospiraceae as a correlate for protection from disease. Using directed microbial culture techniques, we discovered Clostridium immunis, a previously unknown bacterial species from this family, that-when administered to colitis-prone mice-protected them against colitis-associated death. To demonstrate the generalizability of our approach, we used it to identify several commensal organisms that induce intestinal expression of an antimicrobial peptide. Thus, we have used microbe-phenotype triangulation to move beyond the standard correlative microbiome study and identify causal microbes for two completely distinct phenotypes. Identification of disease-modulating commensals by microbe-phenotype triangulation may be more broadly applicable to human microbiome studies.

Read more
September 22, 2019  |  

Differential responses of total and active soil microbial communities to long-term experimental N deposition

Abstract The relationship between total and metabolically active soil microbial communities can provide insight into how these communities are impacted by environmental change, which may impact the flow of energy and cycling of nutrients in the future. For example, the anthropogenic release of biologically available N has dramatically increased over the last 150 years, which can alter the processes controlling C storage in terrestrial ecosystems. In a northern hardwood forest ecosystem located in Michigan, USA, nearly 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. A microbial mechanism underlies this response, as compositional changes in the soil microbial community have been concomitantly documented with these biogeochemical changes. Here, we co-extracted DNA and RNA from decaying leaf litter to determine if experimental atmospheric N deposition has lowered the diversity and altered the composition of the whole communities of bacteria and fungi (i.e., DNA-based) and well as its active members (i.e., RNA-based). In our experiment, experimental N deposition did not affect the composition, diversity, or richness of the total forest floor fungal community, but did lower the diversity (-8%), as well as altered the composition of the active fungal community. In contrast, neither the total nor active forest floor bacterial community was significantly affected by experimental N deposition. Our results suggest that future rates of atmospheric N deposition can fundamentally alter the organization of the saprotrophic soil fungal community, key mediators of C cycling in terrestrial environments.

Read more
September 22, 2019  |  

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

Read more
September 22, 2019  |  

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis.

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (?123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3’UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of pri-miR-BHRF1-2 and 1-3 in the ?123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the ?123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis. Copyright © 2017 Elsevier Inc. All rights reserved.

Read more
September 22, 2019  |  

BIGMAC : breaking inaccurate genomes and merging assembled contigs for long read metagenomic assembly.

The problem of de-novo assembly for metagenomes using only long reads is gaining attention. We study whether post-processing metagenomic assemblies with the original input long reads can result in quality improvement. Previous approaches have focused on pre-processing reads and optimizing assemblers. BIGMAC takes an alternative perspective to focus on the post-processing step.Using both the assembled contigs and original long reads as input, BIGMAC first breaks the contigs at potentially mis-assembled locations and subsequently scaffolds contigs. Our experiments on metagenomes assembled from long reads show that BIGMAC can improve assembly quality by reducing the number of mis-assemblies while maintaining or increasing N50 and N75. Moreover, BIGMAC shows the largest N75 to number of mis-assemblies ratio on all tested datasets when compared to other post-processing tools. BIGMAC demonstrates the effectiveness of the post-processing approach in improving the quality of metagenomic assemblies.

Read more
September 22, 2019  |  

The maize W22 genome provides a foundation for functional genomics and transposon biology.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

Read more
September 22, 2019  |  

SMRT-Cappable-seq reveals complex operon variants in bacteria.

Current methods for genome-wide analysis of gene expression require fragmentation of original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally, in vivo processing of transcripts confounds the accurate identification of the 5′ and 3′ ends of operons. Here we develop a methodology called SMRT-Cappable-seq that combines the isolation of un-fragmented primary transcripts with single-molecule long read sequencing. Applied to E. coli, this technology results in an accurate definition of the transcriptome with 34% of known operons from RegulonDB being extended by at least one gene. Furthermore, 40% of transcription termination sites have read-through that alters the gene content of the operons. As a result, most of the bacterial genes are present in multiple operon variants reminiscent of eukaryotic splicing. By providing such granularity in the operon structure, this study represents an important resource for the study of prokaryotic gene network and regulation.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.