scholarly journals Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Caitlin M. Singleton ◽  
Francesca Petriglieri ◽  
Jannie M. Kristensen ◽  
Rasmus H. Kirkegaard ◽  
Thomas Y. Michaelsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Sequencing Data ◽  
High Quality ◽  
16S Rrna Amplicon Sequencing ◽  
Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

scholarly journals Connecting structure to function with the recovery of over 1000 high-quality activated sludge metagenome-assembled genomes encoding full-length rRNA genes using long-read sequencing

2020 ◽  
Author(s):  
Caitlin M Singleton ◽  
Francesca Petriglieri ◽  
Jannie M Kristensen ◽  
Rasmus H Kirkegaard ◽  
Thomas Y Michaelsen ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Treatment Plant ◽  
Full Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Fluorescence In Situ Hybridisation ◽  
High Quality ◽  
Recovery Processes ◽  
Long Read ◽  
Pollution Removal

AbstractMicroorganisms are critical to water recycling, pollution removal and resource recovery processes in the wastewater industry. While the structure of this complex community is increasingly understood based on 16S rRNA gene studies, this structure cannot currently be linked to functional potential due to the absence of high-quality metagenome-assembled genomes (MAGs) with full-length rRNA genes for nearly all species. Here, we sequence 23 Danish full-scale wastewater treatment plant metagenomes, producing >1 Tbp of long-read and >0.9 Tbp of short-read data. We recovered 1083 high-quality MAGs, including 57 closed circular genomes. The MAGs accounted for ~30% of the community, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We show how novel high-quality MAGs in combination with >13 years of amplicon data, Raman microspectroscopy and fluorescence in situ hybridisation can be used to uncover abundant undescribed lineages belonging to important functional groups.

scholarly journals NanoRTax, a real-time pipeline for taxonomic and diversity analysis of nanopore 16S rRNA amplicon sequencing data

2021 ◽  
Author(s):  
Héctor Rodriguez-Perez ◽  
Laura Ciuffreda ◽  
Carlos Flores
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Amplicon Sequencing ◽  
Sequencing Data ◽  
Real Time Analysis ◽  
16S Rrna Amplicon Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Cost Efficient ◽  
User Friendly

Abstract The study of microbial communities and their applications have been leveraged by the advances in sequencing techniques and bioinformatics tools. The Oxford Nanopore Technologies long read sequencing by nanopores provides a portable and cost-efficient platform for sequencing assays opening the possibility of its application outside specialized environments and real-time analysis of data. To complement the existing efficient library preparation protocol with a streamlined analytic workflow, here we present NanoRTax, a nextflow pipeline for nanopore 16S rRNA amplicon data that features state-of-art taxonomic classification tools and real-time capability. The pipeline is paired with a web-based visual interface to enable user-friendly inspections of the experiment in progress.

scholarly journals Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Benjamin J. Callahan ◽  
Dmitry Grinevich ◽  
Siddhartha Thakur ◽  
Michael A. Balamotis ◽  
Tuval Ben Yehezkel
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Strain Identification ◽  
Long Reads ◽  
Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

scholarly journals Dramatic differences in gut bacterial densities help to explain the relationship between diet and habitat in rainforest ants

2017 ◽  
Author(s):  
Jon G Sanders ◽  
Piotr Lukasik ◽  
Megan E Frederickson ◽  
Jacob A Russell ◽  
Ryuichi Koga ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Diversity ◽  
Tropical Rainforest ◽  
Amplicon Sequencing ◽  
Sequencing Data ◽  
Lowland Tropical Forest ◽  
16S Rrna Amplicon Sequencing ◽  
Microbial Symbionts ◽  
Microbial Symbiosis ◽  
Diversity Profiles

AbstractAbundance is a key parameter in microbial ecology, and important to estimates of potential metabolite flux, impacts of dispersal, and sensitivity of samples to technical biases such as laboratory contamination. However, modern amplicon-based sequencing techniques by themselves typically provide no information about the absolute abundance of microbes. Here, we use fluorescence microscopy and quantitative PCR as independent estimates of microbial abundance to test the hypothesis that microbial symbionts have enabled ants to dominate tropical rainforest canopies by facilitating herbivorous diets, and compare these methods to microbial diversity profiles from 16S rRNA amplicon sequencing. Through a systematic survey of ants from a lowland tropical forest, we show that the density of gut microbiota varies across several orders of magnitude among ant lineages, with median individuals from many genera only marginally above detection limits. Supporting the hypothesis that microbial symbiosis is important to dominance in the canopy, we find that the abundance of gut bacteria is positively correlated with stable isotope proxies of herbivory among canopy-dwelling ants, but not among ground-dwelling ants. Notably, these broad findings are much more evident in the quantitative data than in the 16S rRNA sequencing data. Our results help to resolve a longstanding question in tropical rainforest ecology, and have broad implications for the interpretation of sequence-based surveys of microbial diversity.

scholarly journals Low-dissolved-oxygen nitrification in tropical sewage: an investigation on potential, performance and functional microbial community

2018 ◽  
Vol 77 (9) ◽  
pp. 2274-2283 ◽  
Author(s):  
S. W. How ◽  
S. Y. Lim ◽  
P. B. Lim ◽  
A. M. Aris ◽  
G. C. Ngoh ◽  
...  
Keyword(s):  
Dissolved Oxygen ◽  
Batch Reactor ◽  
In Situ Hybridisation ◽  
Sewage Treatment ◽  
Oxygen Affinity ◽  
Amplicon Sequencing ◽  
Fluorescence In Situ Hybridisation ◽  
Low Dissolved Oxygen ◽  
16S Rrna Amplicon Sequencing ◽  
Low Do

Abstract Intensive aeration for nitrification is a major energy consumer in sewage treatment plants (STPs). Low-dissolved-oxygen (low-DO) nitrification has the potential to lower the aeration demand. However, the applicability of low-DO nitrification in the tropical climate is not well-understood. In this study, the potential of low-DO nitrification in tropical setting was first examined using batch kinetic experiments. Subsequently, the performance of low-DO nitrification was investigated in a laboratory-scale sequential batch reactor (SBR) for 42 days using real tropical sewage. The batch kinetic experiments showed that the seed sludge has a relatively high oxygen affinity. Thus, the rate of nitrification was not significantly reduced at low DO concentrations (0.5 mg/L). During the operation of the low-DO nitrification SBR, 90% of NH4-N was removed. The active low-DO nitrification was mainly attributed to the limited biodegradable organics in the sewage. Fluorescence in-situ hybridisation and 16S rRNA amplicon sequencing revealed the nitrifiers were related to Nitrospira genus and Nitrosomonadaceae family. Phylogenetic analysis suggests 47% of the operational taxonomic units in Nitrospira genus are closely related to a comammox bacteria. This study has demonstrated active low-DO nitrification in tropical setting, which is a more sustainable process that could significantly reduce the energy footprint of STPs.

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

2020 ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

AbstractFreshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on >97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages.

scholarly journals Machine Learning-assisted Identification of Bioindicators Predicts Medium-chain Carboxylate Production Performance of an Anaerobic Mixed Culture

2020 ◽  
Author(s):  
Bin Liu ◽  
Heike Sträuber ◽  
Joao Saraiva ◽  
Hauke Harms ◽  
Sandra Godinho Silva ◽  
...  
Keyword(s):  
Machine Learning ◽  
16S Rrna ◽  
Retention Time ◽  
Hydraulic Retention Time ◽  
Amplicon Sequencing ◽  
Production Performance ◽  
Chain Elongation ◽  
Sequencing Data ◽  
Medium Chain ◽  
16S Rrna Amplicon Sequencing

Abstract Background: The ability to quantitatively predict ecophysiological functions of microbial communities provides an important step to engineer microbiota for desired functions related to specific biochemical conversions. Here, we present the quantitative prediction of medium-chain carboxylate production in two continuous anaerobic bioreactors from 16S rRNA gene dynamics in enrichment cultures. Results: By progressively shortening the hydraulic retention time from 8 days to 2 days with different temporal schemes in both bioreactors operated for 211 days, we achieved higher productivities and yields of the target products n-caproate and n-caprylate. The datasets generated from each bioreactor were applied independently for training and testing in machine learning. A predictive model was generated by employing the random forest algorithm using 16S rRNA amplicon sequencing data. More than 90% accuracy in the prediction of n-caproate and n-caprylate productivities was achieved. Four inferred bioindicators belonging to the genera Olsenella, Lactobacillus, Syntrophococcus and Clostridium IV suggest their relevance to the higher carboxylate productivity at shorter hydraulic retention time. The recovery of metagenome-assembled genomes of these bioindicators confirmed their genetic potential to perform key steps of medium-chain carboxylate production.Conclusions: Shortening the hydraulic retention time of the continuous bioreactor systems allows to shape the communities with desired chain elongation functions. Using machine-learning, we demonstrated that 16S rRNA amplicon sequencing data can be used to predict bioreactor process performance quantitatively and accurately. Characterising and harnessing bioindicators holds promise to manage reactor microbiota towards selection of the target processes. Our mathematical framework is transferrable to other ecosystem processes and 3 microbial systems where community dynamics is linked to key functions. The general methodology can be adapted to data types of other functional categories such as genes, transcripts, proteins or metabolites.

scholarly journals Ultra-accurate Microbial Amplicon Sequencing Directly from Complex Samples with Synthetic Long Reads

2020 ◽  
Author(s):  
Benjamin J Callahan ◽  
Dmitry Grinevich ◽  
Siddhartha Thakur ◽  
Michael A Balamotis ◽  
Tuval Ben Yehezkel
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Genes ◽  
Sequencing Technology ◽  
Complex Samples ◽  
Long Reads ◽  
Long Read

AbstractOut of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing technology. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Here, we describe and analytically validate LoopSeq, a commercially-available synthetic long-read (SLR) sequencing technology that generates highly-accurate long reads from standard short reads. LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq applied to full-length 16S rRNA genes from known strains in a microbial community perfectly recovered the full diversity of full-length exact sequence variants in a known microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kilobases in length. Analysis of rinsate from retail meat samples demonstrated that LoopSeq full-length 16S rRNA synthetic long-reads could accurately classify organisms down to the species level, and could differentiate between different strains within species identified by the CDC as potential foodborne pathogens. The order-of-magnitude improvement in both length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex and low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

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