nf-core/mag: a best-practice pipeline for metagenome hybrid assembly and binning

The analysis of shotgun metagenomic data provides valuable insights into microbial communities, while allowing resolution at individual genome level. In absence of complete reference genomes, this requires the reconstruction of metagenome assembled genomes (MAGs) from sequencing reads. We present the nf-core/mag pipeline for metagenome assembly, binning and taxonomic classification. It can optionally combine short and long reads to increase assembly continuity and utilize sample-wise group-information for co-assembly and genome binning. The pipeline is easy to install - all dependencies are provided within containers -, portable and reproducible. It is written in Nextflow and developed as part of the nf-core initiative for best-practice pipeline development. All code is hosted on GitHub under the nf-core organization https://github.com/nf-core/mag and released under the MIT license.

Download Full-text

An accurate and fast alignment-free method for profiling microbial communities

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720017400017 ◽

2017 ◽

Vol 15 (03) ◽

pp. 1740001 ◽

Cited By ~ 4

Author(s):

Diem-Trang Pham ◽

Shanshan Gao ◽

Vinhthuy Phan

Keyword(s):

Microbial Communities ◽

Accurate Method ◽

Metagenomic Data ◽

Quadratic Programs ◽

Microbial Genomes ◽

Microbial Species ◽

Alignment Free ◽

Human Dna ◽

Computationally Expensive ◽

Reference Genomes

Determining abundances of microbial genomes in metagenomic samples is an important problem in analyzing metagenomic data. Although homology-based methods are popular, they have shown to be computationally expensive due to the alignment of tens of millions of reads from metagenomic samples to reference genomes of hundreds to thousands of environmental microbial species. We introduce an efficient alignment-free approach to estimate abundances of microbial genomes in metagenomic samples. The approach is based on solving linear and quadratic programs, which are represented by genome-specific markers (GSM). We compared our method against popular alignment-free and homology-based methods. Without contamination, our method was more accurate than other alignment-free methods while being much faster than a homology-based method. In more realistic settings where samples were contaminated with human DNA, our method was the most accurate method in predicting abundance at varying levels of contamination. We achieve higher accuracy than both alignment-free and homology-based methods.

Download Full-text

Testing the advantages and disadvantages of short- and long- read eukaryotic metagenomics using simulated reads

10.21203/rs.2.10271/v3 ◽

2020 ◽

Author(s):

William S Pearman ◽

Nikki E Freed ◽

Olin K Silander

Keyword(s):

Microbial Communities ◽

Classification Accuracy ◽

Community Diversity ◽

Read Length ◽

Metagenomic Data ◽

Taxonomic Resolution ◽

Sequence Length ◽

Advantages And Disadvantages ◽

Long Reads ◽

Oxford Nanopore

Abstract Background The first step in understanding ecological community diversity and dynamics is quantifying community membership. An increasingly common method for doing so is through metagenomics. Because of the rapidly increasing popularity of this approach, a large number of computational tools and pipelines are available for analysing metagenomic data. However, the majority of these tools have been designed and benchmarked using highly accurate short read data (i.e. Illumina), with few studies benchmarking classification accuracy for long error-prone reads (PacBio or Oxford Nanopore). In addition, few tools have been benchmarked for non-microbial communities. Results Here we compare simulated long reads from Oxford Nanopore and Pacific Biosciences with high accuracy Illumina read sets to systematically investigate the effects of sequence length and taxon type on classification accuracy for metagenomic data from both microbial and non-microbial communities. We show that very generally, classification accuracy is far lower for non-microbial communities, even at low taxonomic resolution (e.g. family rather than genus). We then show that for two popular taxonomic classifiers, long reads can significantly increase classification accuracy, and this is most pronounced for non-microbial communities. Conclusions This work provides insight on the expected accuracy for metagenomic analyses for different taxonomic groups, and establishes the point at which read length becomes more important than error rate for assigning the correct taxon.

Download Full-text

Testing the advantages and disadvantages of short- and long- read eukaryotic metagenomics using simulated reads

10.21203/rs.2.10271/v4 ◽

2020 ◽

Author(s):

William S Pearman ◽

Nikki E Freed ◽

Olin K Silander

Keyword(s):

Microbial Communities ◽

Classification Accuracy ◽

Community Diversity ◽

Read Length ◽

Metagenomic Data ◽

Taxonomic Resolution ◽

Sequence Length ◽

Advantages And Disadvantages ◽

Long Reads ◽

Oxford Nanopore

Abstract Background: The first step in understanding ecological community diversity and dynamics is quantifying community membership. An increasingly common method for doing so is through metagenomics. Because of the rapidly increasing popularity of this approach, a large number of computational tools and pipelines are available for analysing metagenomic data. However, the majority of these tools have been designed and benchmarked using highly accurate short read data (i.e. Illumina), with few studies benchmarking classification accuracy for long error-prone reads (PacBio or Oxford Nanopore). In addition, few tools have been benchmarked for non-microbial communities. Results: Here we compare simulated long reads from Oxford Nanopore and Pacific Biosciences with high accuracy Illumina read sets to systematically investigate the effects of sequence length and taxon type on classification accuracy for metagenomic data from both microbial and non-microbial communities. We show that very generally, classification accuracy is far lower for non-microbial communities, even at low taxonomic resolution (e.g. family rather than genus). We then show that for two popular taxonomic classifiers, long reads can significantly increase classification accuracy, and this is most pronounced for non-microbial communities.Conclusions: This work provides insight on the expected accuracy for metagenomic analyses for different taxonomic groups, and establishes the point at which read length becomes more important than error rate for assigning the correct taxon.

Download Full-text

Benchmarking hybrid assembly approaches for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing

BMC Genomics ◽

10.1186/s12864-020-07041-8 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Zhao Chen ◽

David L. Erickson ◽

Jianghong Meng

Keyword(s):

Virulence Genes ◽

Reference Genome ◽

Bacterial Pathogens ◽

Nanopore Sequencing ◽

Hybrid Assembly ◽

Pan Genome ◽

Long Reads ◽

Oxford Nanopore ◽

Hybrid Assemblies ◽

Reference Genomes

Abstract Background We benchmarked the hybrid assembly approaches of MaSuRCA, SPAdes, and Unicycler for bacterial pathogens using Illumina and Oxford Nanopore sequencing by determining genome completeness and accuracy, antimicrobial resistance (AMR), virulence potential, multilocus sequence typing (MLST), phylogeny, and pan genome. Ten bacterial species (10 strains) were tested for simulated reads of both mediocre- and low-quality, whereas 11 bacterial species (12 strains) were tested for real reads. Results Unicycler performed the best for achieving contiguous genomes, closely followed by MaSuRCA, while all SPAdes assemblies were incomplete. MaSuRCA was less tolerant of low-quality long reads than SPAdes and Unicycler. The hybrid assemblies of five antimicrobial-resistant strains with simulated reads provided consistent AMR genotypes with the reference genomes. The MaSuRCA assembly of Staphylococcus aureus with real reads contained msr(A) and tet(K), while the reference genome and SPAdes and Unicycler assemblies harbored blaZ. The AMR genotypes of the reference genomes and hybrid assemblies were consistent for the other five antimicrobial-resistant strains with real reads. The numbers of virulence genes in all hybrid assemblies were similar to those of the reference genomes, irrespective of simulated or real reads. Only one exception existed that the reference genome and hybrid assemblies of Pseudomonas aeruginosa with mediocre-quality long reads carried 241 virulence genes, whereas 184 virulence genes were identified in the hybrid assemblies of low-quality long reads. The MaSuRCA assemblies of Escherichia coli O157:H7 and Salmonella Typhimurium with mediocre-quality long reads contained 126 and 118 virulence genes, respectively, while 110 and 107 virulence genes were detected in their MaSuRCA assemblies of low-quality long reads, respectively. All approaches performed well in our MLST and phylogenetic analyses. The pan genomes of the hybrid assemblies of S. Typhimurium with mediocre-quality long reads were similar to that of the reference genome, while SPAdes and Unicycler were more tolerant of low-quality long reads than MaSuRCA for the pan-genome analysis. All approaches functioned well in the pan-genome analysis of Campylobacter jejuni with real reads. Conclusions Our research demonstrates the hybrid assembly pipeline of Unicycler as a superior approach for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.

Download Full-text

Complete assembly of parental haplotypes with trio binning

10.1101/271486 ◽

2018 ◽

Cited By ~ 8

Author(s):

Sergey Koren ◽

Arang Rhie ◽

Brian P. Walenz ◽

Alexander T. Dilthey ◽

Derek M. Bickhart ◽

...

Keyword(s):

Best Practice ◽

Bos Taurus ◽

New Approach ◽

Haplotype Variation ◽

Long Reads ◽

Bos Taurus Indicus ◽

Alternative Approaches ◽

Complex Structural ◽

Reference Genomes

AbstractReference genome projects have historically selected inbred individuals to minimize heterozygosity and simplify assembly. We challenge this dogma and present a new approach designed specifically for heterozygous genomes. “Trio binning” uses short reads from two parental genomes to partition long reads from an offspring into haplotype-specific sets prior to assembly. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. On a benchmark human trio, this method achieved high accuracy and recovered complex structural variants missed by alternative approaches. To demonstrate its effectiveness on a heterozygous genome, we sequenced an F1 cross between cattle subspecies Bos taurus taurus and Bos taurus indicus, and completely assembled both parental haplotypes with NG50 haplotig sizes >20 Mbp and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We propose trio binning as a new best practice for diploid genome assembly that will enable new studies of haplotype variation and inheritance.

Download Full-text

Testing the advantages and disadvantages of short- and long- read eukaryotic metagenomics using simulated reads

10.21203/rs.2.10271/v2 ◽

2019 ◽

Author(s):

William S Pearman ◽

Nikki E Freed ◽

Olin K Silander

Keyword(s):

Microbial Communities ◽

Classification Accuracy ◽

Community Diversity ◽

Read Length ◽

Metagenomic Data ◽

Taxonomic Resolution ◽

Sequence Length ◽

Advantages And Disadvantages ◽

Long Reads ◽

Oxford Nanopore

Abstract Background The first step in understanding ecological community diversity and dynamics is quantifying community membership. An increasingly common method for doing so is through metagenomics. Because of the rapidly increasing popularity of this approach, a large number of computational tools and pipelines are available for analysing metagenomic data. However, the majority of these tools have been designed and benchmarked using highly accurate short read data (i.e. Illumina), with few studies benchmarking classification accuracy for long error-prone reads (PacBio or Oxford Nanopore). In addition, few tools have been benchmarked for non-microbial communities.Results Here we compare simulated long reads from Oxford Nanopore and Pacific Biosciences with high accuracy Illumina read sets to systematically investigate the effects of sequence length and taxon type on classification accuracy for metagenomic data from both microbial and non-microbial communities. We show that very generally, classification accuracy is far lower for non-microbial communities, even at low taxonomic resolution (e.g. family rather than genus). We then show that for two popular taxonomic classifiers, long reads can significantly increase classification accuracy, and this is most pronounced for non-microbial communities.Conclusions This work provides insight on the expected accuracy for metagenomic analyses for different taxonomic groups, and establishes the point at which read length becomes more important than error rate for assigning the correct taxon.

Download Full-text

Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan

Scientific Reports ◽

10.1038/s41598-021-91615-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kazutoshi Yoshitake ◽

Gaku Kimura ◽

Tomoko Sakami ◽

Tsuyoshi Watanabe ◽

Yukiko Taniuchi ◽

...

Keyword(s):

Time Series ◽

Microbial Communities ◽

Pacific Coast ◽

Three Dimensional ◽

Amplicon Sequencing ◽

Metagenomic Data ◽

Shotgun Metagenomics ◽

Functional Features ◽

Pacific Coast Of Japan ◽

Marine Microbial Communities

AbstractAlthough numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data (http://marine-meta.healthscience.sci.waseda.ac.jp/omd/), which provides a three-dimensional bird’s-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

Download Full-text

Microbial drivers of methane emissions from unrestored industrial salt ponds

The ISME Journal ◽

10.1038/s41396-021-01067-w ◽

2021 ◽

Author(s):

Jinglie Zhou ◽

Susanna M. Theroux ◽

Clifton P. Bueno de Mesquita ◽

Wyatt H. Hartman ◽

Ye Tian ◽

...

Keyword(s):

Microbial Communities ◽

Methane Flux ◽

Methane Emissions ◽

Metagenomic Data ◽

Rrna Gene ◽

Salt Ponds ◽

Salt Pond ◽

Reference Wetlands ◽

Reference Wetland ◽

The Impact

AbstractWetlands are important carbon (C) sinks, yet many have been destroyed and converted to other uses over the past few centuries, including industrial salt making. A renewed focus on wetland ecosystem services (e.g., flood control, and habitat) has resulted in numerous restoration efforts whose effect on microbial communities is largely unexplored. We investigated the impact of restoration on microbial community composition, metabolic functional potential, and methane flux by analyzing sediment cores from two unrestored former industrial salt ponds, a restored former industrial salt pond, and a reference wetland. We observed elevated methane emissions from unrestored salt ponds compared to the restored and reference wetlands, which was positively correlated with salinity and sulfate across all samples. 16S rRNA gene amplicon and shotgun metagenomic data revealed that the restored salt pond harbored communities more phylogenetically and functionally similar to the reference wetland than to unrestored ponds. Archaeal methanogenesis genes were positively correlated with methane flux, as were genes encoding enzymes for bacterial methylphosphonate degradation, suggesting methane is generated both from bacterial methylphosphonate degradation and archaeal methanogenesis in these sites. These observations demonstrate that restoration effectively converted industrial salt pond microbial communities back to compositions more similar to reference wetlands and lowered salinities, sulfate concentrations, and methane emissions.

Download Full-text

The Microbiome Modeling Toolbox: from microbial interactions to personalized microbial communities

Bioinformatics ◽

10.1093/bioinformatics/bty941 ◽

2018 ◽

Vol 35 (13) ◽

pp. 2332-2334 ◽

Cited By ~ 16

Author(s):

Federico Baldini ◽

Almut Heinken ◽

Laurent Heirendt ◽

Stefania Magnusdottir ◽

Ronan M T Fleming ◽

...

Keyword(s):

Microbial Communities ◽

Microbial Interactions ◽

Microbial Genome ◽

Metagenomic Data ◽

Metabolic Interactions ◽

Constraint Based Modeling ◽

Genome Scale

Abstract Motivation The application of constraint-based modeling to functionally analyze metagenomic data has been limited so far, partially due to the absence of suitable toolboxes. Results To address this gap, we created a comprehensive toolbox to model (i) microbe–microbe and host–microbe metabolic interactions, and (ii) microbial communities using microbial genome-scale metabolic reconstructions and metagenomic data. The Microbiome Modeling Toolbox extends the functionality of the constraint-based reconstruction and analysis toolbox. Availability and implementation The Microbiome Modeling Toolbox and the tutorials at https://git.io/microbiomeModelingToolbox.

Download Full-text

Oral Spirochetes Implicated in Dental Diseases Are Widespread in Normal Human Subjects and Carry Extremely Diverse Integron Gene Cassettes

Applied and Environmental Microbiology ◽

10.1128/aem.00564-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5288-5296 ◽

Cited By ~ 17

Author(s):

Yu-Wei Wu ◽

Mina Rho ◽

Thomas G. Doak ◽

Yuzhen Ye

Keyword(s):

Microbial Communities ◽

De Novo ◽

Human Subjects ◽

Human Microbiome ◽

Metagenomic Data ◽

De Bruijn Graph ◽

Content Type ◽

Gene Cassettes ◽

Dental Diseases ◽

Normal Human

ABSTRACTThe NIH Human Microbiome Project (HMP) has produced several hundred metagenomic data sets, allowing studies of the many functional elements in human-associated microbial communities. Here, we survey the distribution of oral spirochetes implicated in dental diseases in normal human individuals, using recombination sites associated with the chromosomal integron inTreponemagenomes, taking advantage of the multiple copies of the integron recombination sites (repeats) in the genomes, and using a targeted assembly approach that we have developed. We find that integron-containingTreponemaspecies are present in ∼80% of the normal human subjects included in the HMP. Further, we are able tode novoassemble the integron gene cassettes using our constrained assembly approach, which employs a unique application of the de Bruijn graph assembly information; most of these cassette genes were not assembled in whole-metagenome assemblies and could not be identified by mapping sequencing reads onto the known referenceTreponemagenomes due to the dynamic nature of integron gene cassettes. Our study significantly enriches the gene pool known to be carried byTreponemachromosomal integrons, totaling 826 (598 97% nonredundant) genes. We characterize the functions of these gene cassettes: many of these genes have unknown functions. The integron gene cassette arrays found in the human microbiome are extraordinarily dynamic, with different microbial communities sharing only a small number of common genes.

Download Full-text