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Sunday, September 22, 2019

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4).

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. Structural annotation is followed by assignment of protein product names and functions.

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Sunday, September 22, 2019

A near complete snapshot of the Zea mays seedling transcriptome revealed from ultra-deep sequencing.

RNA-sequencing (RNA-seq) enables in-depth exploration of transcriptomes, but typical sequencing depth often limits its comprehensiveness. In this study, we generated nearly 3 billion RNA-Seq reads, totaling 341 Gb of sequence, from a Zea mays seedling sample. At this depth, a near complete snapshot of the transcriptome was observed consisting of over 90% of the annotated transcripts, including lowly expressed transcription factors. A novel hybrid strategy combining de novo and reference-based assemblies yielded a transcriptome consisting of 126,708 transcripts with 88% of expressed known genes assembled to full-length. We improved current annotations by adding 4,842 previously unannotated transcript variants and many…

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Sunday, 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…

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Sunday, September 22, 2019

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes.

Pan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences’ (PacBio’s) long-read technology in particular suffers from high error rates per base. Herein, we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus…

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Sunday, September 22, 2019

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi.

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long…

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Sunday, September 22, 2019

ALE: a generic assembly likelihood evaluation framework for assessing the accuracy of genome and metagenome assemblies.

Researchers need general purpose methods for objectively evaluating the accuracy of single and metagenome assemblies and for automatically detecting any errors they may contain. Current methods do not fully meet this need because they require a reference, only consider one of the many aspects of assembly quality or lack statistical justification, and none are designed to evaluate metagenome assemblies.In this article, we present an Assembly Likelihood Evaluation (ALE) framework that overcomes these limitations, systematically evaluating the accuracy of an assembly in a reference-independent manner using rigorous statistical methods. This framework is comprehensive, and integrates read quality, mate pair orientation and…

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Sunday, September 22, 2019

Widespread polycistronic transcripts in fungi revealed by single-molecule mRNA sequencing.

Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, and Gloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of…

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Sunday, 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…

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Sunday, 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…

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Sunday, September 22, 2019

Genomic and metabolic diversity of Marine Group I Thaumarchaeota in the mesopelagic of two subtropical gyres.

Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and…

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Sunday, September 22, 2019

In vitro characterization of phenylacetate decarboxylase, a novel enzyme catalyzing toluene biosynthesis in an anaerobic microbial community.

Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in…

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Sunday, September 22, 2019

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection.

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut…

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Sunday, September 22, 2019

Next generation sequencing data of a defined microbial mock community.

Generating sequence data of a defined community composed of organisms with complete reference genomes is indispensable for the benchmarking of new genome sequence analysis methods, including assembly and binning tools. Moreover the validation of new sequencing library protocols and platforms to assess critical components such as sequencing errors and biases relies on such datasets. We here report the next generation metagenomic sequence data of a defined mock community (Mock Bacteria ARchaea Community; MBARC-26), composed of 23 bacterial and 3 archaeal strains with finished genomes. These strains span 10 phyla and 14 classes, a range of GC contents, genome sizes, repeat…

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Sunday, September 22, 2019

PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: Insights, challenges andopportunities.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored…

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Sunday, September 22, 2019

Discovery of enzymes for toluene synthesis from anoxic microbial communities.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This…

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