EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data

Miller, Christopher S.; Baker, Brett J.; Thomas, Brian C.; Singer, Steven W.; Banfield, Jillian F.

doi:10.1186/gb-2011-12-5-r44

Cited by 325 publications

(377 citation statements)

References 51 publications

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“…11), as genomic bins were not recovered for all these taxa. To verify the accuracy of our binned genomes, near-full-length ribosomal 16S rRNA gene sequences were reconstructed from unassembled Illumina reads from Marcellus fluids and an Utica fluid GB enrichment culture using EMIRGE (Supplementary Data File 1) 11 . To reconstruct 16S rRNA gene sequences we followed the protocol with trimmed paired-end reads where both reads were at least 20 nucleotides used as inputs and 50 iterations.…”

Section: Methodsmentioning

confidence: 99%

“…A high percentage of sequenced reads mapped to the assemblies (89-99%) (Supplementary Table 3), signifying that the underlying data were well represented. We also validated that the assembled genomes reflected the microbial identities and abundances in the unassembled reads, by comparing genome bin relative abundance to reconstructed near-full-length 16S rRNA genes 11 (Supplementary Data File 1).…”

Section: Reconstruction Of Persisting Shale Genomesmentioning

confidence: 99%

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Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

et al. 2016

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Hydraulic fracturing is the industry standard for extracting hydrocarbons from shale formations. Attention has been paid to the economic benefits and environmental impacts of this process, yet the biogeochemical changes induced in the deep subsurface are poorly understood. Recent single-gene investigations revealed that halotolerant microbial communities were enriched after hydraulic fracturing. Here, the reconstruction of 31 unique genomes coupled to metabolite data from the Marcellus and Utica shales revealed that many of the persisting organisms play roles in methylamine cycling, ultimately supporting methanogenesis in the deep biosphere. Fermentation of injected chemical additives also sustains long-term microbial persistence, while thiosulfate reduction could produce sulfide, contributing to reservoir souring and infrastructure corrosion. Extensive links between viruses and microbial hosts demonstrate active viral predation, which may contribute to the release of labile cellular constituents into the extracellular environment. Our analyses show that hydraulic fracturing provides the organismal and chemical inputs for colonization and persistence in the deep terrestrial subsurface. S hale gas accounts for one-third of natural gas energy resources worldwide. It has been estimated that shale gas will provide half of the natural gas in the USA, annually, by 2040, with the Marcellus shale in the Appalachian Basin projected to produce three times more than any other formation 1 . Recovery of these hydrocarbons is dependent on hydraulic fracturing technologies, where the high-pressure injection of water and chemical additives generates extensive fractures in the shale matrix. Hydrocarbons trapped in tiny pore spaces are subsequently released and collected at the wellpad surface, together with a portion of the injected fluids that have reacted with the shale formation. The mixture of injected fluids and hydrocarbons collected is referred to as 'produced fluids'.Microbial metabolism and growth in hydrocarbon reservoirs has both positive and negative impacts on energy recovery. Whereas stimulation of methanogens in coal beds enhances energy recovery 2 , bacterial hydrogen sulfide production ('reservoir souring') decreases profits and contributes to corrosion and the risk of environmental contamination 3 . Additionally, biomass accumulation within newly generated fractures may reduce their permeability, decreasing natural gas recovery. Despite these potential microbial impacts, little is known about the function and activity of microorganisms in hydraulically fractured shale.Initial work by our group and others 4-9 used single marker gene analyses to identify microorganisms from several geographically distinct shale formations. These analyses showed similar halotolerant taxa in produced fluids several months after hydraulic fracturing. To assign functional roles to these organisms, we conducted metagenomic and metabolite analyses on input and produced fluids up to a year after hydraulic fracturing (HF) from two Appala...

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Section: Methodsmentioning

confidence: 99%

Section: Reconstruction Of Persisting Shale Genomesmentioning

confidence: 99%

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

et al. 2016

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“…Sequencing reads belonging to the abundant Leptospirillum groups II and III rRNA genes were removed from the ribosomal RNA data files by mapping reads using Bowtie (Langmead, 2010). SSU rRNA gene sequences were then reconstructed from the remainder Leptospirillum-subtracted reads using EMIRGE (Miller et al, 2011) with parameters -l 101 -i 300 -s 100 -phred33, and run until 40 iterations. Sequences classified as chimeras by uchime (Edgar et al, 2011) and DECIPHER (Wright et al, 2012) were excluded.…”

Section: Methodsmentioning

confidence: 99%

Community transcriptomics reveals unexpected high microbial diversity in acidophilic biofilm communities

et al. 2014

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A fundamental question in microbial ecology relates to community structure, and how this varies across environment types. It is widely believed that some environments, such as those at very low pH, host simple communities based on the low number of taxa, possibly due to the extreme environmental conditions. However, most analyses of species richness have relied on methods that provide relatively low ribosomal RNA (rRNA) sampling depth. Here we used community transcriptomics to analyze the microbial diversity of natural acid mine drainage biofilms from the Richmond Mine at Iron Mountain, California. Our analyses target deep pools of rRNA gene transcripts recovered from both natural and laboratory-grown biofilms across varying developmental stages. In all, 91.8% of the B254 million Illumina reads mapped to rRNA genes represented in the SILVA database. Up to 159 different taxa, including Bacteria, Archaea and Eukaryotes, were identified. Diversity measures, ordination and hierarchical clustering separate environmental from laboratory-grown biofilms. In part, this is due to the much larger number of rare members in the environmental biofilms. Although Leptospirillum bacteria generally dominate biofilms, we detect a wide variety of other Nitrospira organisms present at very low abundance. Bacteria from the Chloroflexi phylum were also detected. The results indicate that the primary characteristic that has enabled prior extensive cultivation-independent 'omic' analyses is not simplicity but rather the high dominance by a few taxa. We conclude that a much larger variety of organisms than previously thought have adapted to this extreme environment, although only few are selected for at any one time.

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“…Given that virtually all environments where life exists can now be probed by metagenomic methods, we anticipate that the combined computational-experimental approach will greatly expand the diversity of known CRISPRCas systems, enabling new technologies for biological research and clinical applications. For the AMD data, DNA extraction methods and short read sequencing were reported by Denef and Banfield (2012) 36 and Miller et al (2011) 37 . For the Rifle data, DNA extraction, sequencing, assembly, and genome reconstruction were described by Anantharaman et al (2016) 26 and Brown et al (2015) 6 .…”

mentioning

confidence: 99%

New CRISPR–Cas systems from uncultivated microbes

Burstein

Harrington

Strutt

et al. 2016

Nature

497

369

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CRISPR-Cas systems provide microbes with adaptive immunity by employing short sequences, termed spacers, that guide Cas proteins to cleave foreign DNA 1,2 . Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA recognizes and cleaves targeted sequences 3,4 . The programmable nature of these minimal systems has enabled their repurposing as a versatile technology that is broadly revolutionizing biological and clinical research 5 . However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving untapped the vast majority of enzymes from organisms that have not been cultured. Metagenomics, the sequencing of DNA extracted from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms 6,7 . Here, using genome-resolved metagenomics, we identified novel CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in littlestudied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two

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EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data

Cited by 325 publications

References 51 publications

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

Community transcriptomics reveals unexpected high microbial diversity in acidophilic biofilm communities

New CRISPR–Cas systems from uncultivated microbes

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