scholarly journals Metagenomic methylation patterns resolve complex microbial genomes

2021 ◽  
Author(s):  
Elizabeth G. Wilbanks ◽  
Hugo Doré ◽  
Meredith H. Ashby ◽  
Cheryl Heiner ◽  
Richard J. Roberts ◽  
...  
Keyword(s):  
Evolutionary Dynamics ◽  
Bacterial Genome ◽  
Dna Methyltransferases ◽  
Microbial Consortia ◽  
Bacterial Genomes ◽  
Genomic Change ◽  
Long Read ◽  
Dominant Bacteria ◽  
Methylation Patterns ◽  
Restriction Modification

AbstractThe plasticity of bacterial and archaeal genomes makes examining their ecological and evolutionary dynamics both exciting and challenging. The same mechanisms that enable rapid genomic change and adaptation confound current approaches for recovering complete genomes from metagenomes. Here, we use strain-specific patterns of DNA methylation to resolve complex bacterial genomes from the long-read metagenome of a marine microbial consortia, the “pink berries” of the Sippewissett Marsh. Unique combinations of restriction-modification (RM) systems encoded by the bacteria produced distinctive methylation profiles that accurately binned and classified metagenomic sequences. We linked the methylation patterns of each metagenome-assembled genome with encoded DNA methyltransferases and discovered new restriction modification (RM) defense systems, including novel associations of RM systems with RNase toxins. Using this approach, we finished the largest and most complex circularized bacterial genome ever recovered from a metagenome (7.9 Mb with >600 IS elements), the finished genome of Thiohalocapsa sp. PB-PSB1 the dominant bacteria in the consortia. From these methylation-binned genomes, we identified instances of lateral gene transfer between sulfur-cycling symbionts (Thiohalocapsa sp. PB-PSB1 and Desulfofustis sp. PB-SRB1), phage infection, and strain-level structural variation.

Graph analysis of fragmented long-read bacterial genome assemblies

2019 ◽  
Vol 35 (21) ◽  
pp. 4239-4246 ◽  
Author(s):  
Pierre Marijon ◽  
Rayan Chikhi ◽  
Jean-Stéphane Varré
Keyword(s):  
Simple Procedure ◽  
Bacterial Genome ◽  
Careful Analysis ◽  
Supplementary Information ◽  
Hamiltonian Cycles ◽  
Computational Techniques ◽  
Bacterial Genomes ◽  
Sequencing Project ◽  
Long Read ◽  
Genome Assemblies

Abstract Motivation Long-read genome assembly tools are expected to reconstruct bacterial genomes nearly perfectly; however, they still produce fragmented assemblies in some cases. It would be beneficial to understand whether these cases are intrinsically impossible to resolve, or if assemblers are at fault, implying that genomes could be refined or even finished with little to no additional experimental cost. Results We propose a set of computational techniques to assist inspection of fragmented bacterial genome assemblies, through careful analysis of assembly graphs. By finding paths of overlapping raw reads between pairs of contigs, we recover potential short-range connections between contigs that were lost during the assembly process. We show that our procedure recovers 45% of missing contig adjacencies in fragmented Canu assemblies, on samples from the NCTC bacterial sequencing project. We also observe that a simple procedure based on enumerating weighted Hamiltonian cycles can suggest likely contig orderings. In our tests, the correct contig order is ranked first in half of the cases and within the top-three predictions in nearly all evaluated cases, providing a direction for finishing fragmented long-read assemblies. Availability and implementation https://gitlab.inria.fr/pmarijon/knot . Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals MicroPIPE: An end-to-end solution for high-quality complete bacterial genome construction

2021 ◽  
Author(s):  
Valentine Murigneux ◽  
Leah W. Roberts ◽  
Brian M. Forde ◽  
Minh-Duy Phan ◽  
Nguyen Thi Khanh Nhu ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Sequence Type ◽  
Bacterial Isolates ◽  
Bacterial Genomes ◽  
High Quality ◽  
Oxford Nanopore ◽  
Long Read ◽  
Complete Bacterial Genome ◽  
Assembly Pipeline ◽  
End To End

AbstractOxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers; however, easy and automated construction of high-quality bacterial genomes remains challenging. Here we present MicroPIPE: a reproducible end-to-end bacterial genome assembly pipeline for ONT and Illumina sequencing. To construct MicroPIPE, we evaluated the performance of several tools for genome reconstruction and assessed overall genome accuracy using ONT both natively and with Illumina. Further validation of MicroPIPE was carried out using 11 sequence type (ST)131 Escherichia coli and eight publicly available Gram-negative and Gram-positive bacterial isolates. MicroPIPE uses Singularity containers and the workflow manager Nextflow and is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe.

scholarly journals Trycycler: consensus long-read assemblies for bacterial genomes

2021 ◽  
Author(s):  
Ryan R Wick ◽  
Louise M Judd ◽  
Louise T Cerdeira ◽  
Jane Hawkey ◽  
Guillaume Meric ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Bacterial Genomes ◽  
Manual Intervention ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Multiple Input ◽  
Long Read ◽  
Complete Bacterial Genome ◽  
Genome Assemblies ◽  
Reference Genomes

Assembly of bacterial genomes from long-read data (generated by Oxford Nanopore or Pacific Biosciences platforms) can often be complete: a single contig for each chromosome or plasmid in the genome. However, even complete bacterial genome assemblies constructed solely from long reads still contain a variety of errors, and different assemblies of the same genome often contain different errors. Here, we present Trycycler, a tool which produces a consensus assembly from multiple input assemblies of the same genome. Benchmarking using both simulated and real sequencing reads showed that Trycycler consensus assemblies contained fewer errors than any of those constructed with a single long-read assembler. Post-assembly polishing with Medaka and Pilon further reduced errors and yielded the most accurate genome assemblies in our study. As Trycycler can require human judgement and manual intervention, its output is not deterministic, and different users can produce different Trycycler assemblies from the same input data. However, we demonstrated that multiple users with minimal training converge on similar assemblies that are consistently more accurate than those produced by automated assembly tools. We therefore recommend Trycycler+Medaka+Pilon as an ideal approach for generating high-quality bacterial reference genomes.

scholarly journals Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes

2019 ◽  
Author(s):  
Nicola De Maio ◽  
Liam P. Shaw ◽  
Alasdair Hubbard ◽  
Sophie George ◽  
Nick Sanderson ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Hybrid Assembly ◽  
Bacterial Genomes ◽  
Short Read ◽  
Short Reads ◽  
Genome Reconstruction ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sequencing Platforms

ABSTRACTIllumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods impact on assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or from SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the Enterobacteriaceae family, as these frequently have highly plastic, repetitive genetic structures and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies. Both strategies facilitate high-quality genome reconstruction. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.IMPACT STATEMENTIllumina short-read sequencing is frequently used for tasks in bacterial genomics, such as assessing which species are present within samples, checking if specific genes of interest are present within individual isolates, and reconstructing the evolutionary relationships between strains. However, while short-read sequencing can reveal significant detail about the genomic content of bacterial isolates, it is often insufficient for assessing genomic structure: how different genes are arranged within genomes, and particularly which genes are on plasmids – potentially highly mobile components of the genome frequently carrying antimicrobial resistance elements. This is because Illumina short reads are typically too short to span repetitive structures in the genome, making it impossible to accurately reconstruct these repetitive regions. One solution is to complement Illumina short reads with long reads generated with SMRT Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) sequencing platforms. Using this approach, called ‘hybrid assembly’, we show that we can automatically fully reconstruct complex bacterial genomes of Enterobacteriaceae isolates in the majority of cases (best-performing method: 17/20 isolates). In particular, by comparing different methods we find that using the assembler Unicycler with Illumina and ONT reads represents a low-cost, high-quality approach for reconstructing bacterial genomes using publicly available software.DATA SUMMARYRaw sequencing data and assemblies have been deposited in NCBI under BioProject Accession PRJNA422511 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA422511). We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

scholarly journals MicroPIPE: validating an end-to-end workflow for high-quality complete bacterial genome construction

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Valentine Murigneux ◽  
Leah W. Roberts ◽  
Brian M. Forde ◽  
Minh-Duy Phan ◽  
Nguyen Thi Khanh Nhu ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Easy Access ◽  
Bacterial Genomes ◽  
High Quality ◽  
Manual Intervention ◽  
Complete Genomes ◽  
Long Read ◽  
Complete Bacterial Genome ◽  
Assembly Pipeline ◽  
End To End

Abstract Background Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers due to the accessibility and affordability of its devices. However, easy and automated construction of high-quality bacterial genomes using nanopore reads remains challenging. Here we aimed to create a reproducible end-to-end bacterial genome assembly pipeline using ONT in combination with Illumina sequencing. Results We evaluated the performance of several popular tools used during genome reconstruction, including base-calling, filtering, assembly, and polishing. We also assessed overall genome accuracy using ONT both natively and with Illumina. All steps were validated using the high-quality complete reference genome for the Escherichia coli sequence type (ST)131 strain EC958. Software chosen at each stage were incorporated into our final pipeline, MicroPIPE. Further validation of MicroPIPE was carried out using 11 additional ST131 E. coli isolates, which demonstrated that complete circularised chromosomes and plasmids could be achieved without manual intervention. Twelve publicly available Gram-negative and Gram-positive bacterial genomes (with available raw ONT data and matched complete genomes) were also assembled using MicroPIPE. We found that revised basecalling and updated assembly of the majority of these genomes resulted in improved accuracy compared to the current publicly available complete genomes. Conclusions MicroPIPE is built in modules using Singularity container images and the bioinformatics workflow manager Nextflow, allowing changes and adjustments to be made in response to future tool development. Overall, MicroPIPE provides an easy-access, end-to-end solution for attaining high-quality bacterial genomes. MicroPIPE is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe.

scholarly journals Comparison of De Novo Assembly Strategies for Bacterial Genomes

2021 ◽  
Vol 22 (14) ◽  
pp. 7668
Author(s):  
Pengfei Zhang ◽  
Dike Jiang ◽  
Yin Wang ◽  
Xueping Yao ◽  
Yan Luo ◽  
...  
Keyword(s):  
De Novo ◽  
Bacterial Genome ◽  
Sequencing Data ◽  
Bacterial Genomes ◽  
Accurate Analysis ◽  
Short Read ◽  
Sequencing Errors ◽  
Assembly Accuracy ◽  
Protein Prediction ◽  
Long Read

(1) Background: Short-read sequencing allows for the rapid and accurate analysis of the whole bacterial genome but does not usually enable complete genome assembly. Long-read sequencing greatly assists with the resolution of complex bacterial genomes, particularly when combined with short-read Illumina data. However, it is not clear how different assembly strategies affect genomic accuracy, completeness, and protein prediction. (2) Methods: we compare different assembly strategies for Haemophilus parasuis, which causes Glässer’s disease, characterized by fibrinous polyserositis and arthritis, in swine by using Illumina sequencing and long reads from the sequencing platforms of either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio). (3) Results: Assembly with either PacBio or ONT reads, followed by polishing with Illumina reads, facilitated high-quality genome reconstruction and was superior to the long-read-only assembly and hybrid-assembly strategies when evaluated in terms of accuracy and completeness. An equally excellent method was correction with Homopolish after the ONT-only assembly, which had the advantage of avoiding hybrid sequencing with Illumina. Furthermore, by aligning transcripts to assembled genomes and their predicted CDSs, the sequencing errors of the ONT assembly were mainly indels that were generated when homopolymer regions were sequenced, thus critically affecting protein prediction. Polishing can fill indels and correct mistakes. (4) Conclusions: The assembly of bacterial genomes can be directly achieved by using long-read sequencing techniques. To maximize assembly accuracy, it is essential to polish the assembly with homologous sequences of related genomes or sequencing data from short-read technology.

scholarly journals Do You Want to Build a Genome? Benchmarking Hybrid Bacterial Genome Assembly Methods

2021 ◽  
Author(s):  
Georgia L Breckell ◽  
Olin K Silander
Keyword(s):  
Genome Assembly ◽  
Bacterial Genome ◽  
Ground Truth ◽  
Bacterial Genomes ◽  
Short Read ◽  
Assembly Method ◽  
Assembly Accuracy ◽  
A Genome ◽  
Lower Accuracy ◽  
Long Read

Long read sequencing technologies now allow routine highly contiguous assembly of bacterial genomes. However, because of the lower accuracy of some long read data, it is often combined with short read data (e.g. Illumina), to improve assembly quality. There are a number of methods available for producing such hybrid assemblies. Here we use Illumina and Oxford Nanopore (ONT) data from 49 natural isolates of Escherichia coli to characterise differences in assembly accuracy for five assembly methods (Canu, Unicycler, Raven, Flye, and Redbean). We evaluate assembly accuracy using five metrics designed to measure structural accuracy and sequence accuracy (indel and substitution frequency). We assess structural accuracy by quantifying (1) the contiguity of chromosomes and plasmids; (2) the fraction of concordantly mapped Illumina reads withheld from the assembly; and (3) whether rRNA operons are correctly oriented. We assess indel and substitution frequency by quantifying (1) the fraction of open reading frames that appear truncated and (2) the number of variants that are called using Illumina reads only. Applying these assembly metrics to a large number of E. coli strains, we find that different assembly methods offer different advantages. In particular, we find that Unicycler assemblies have the highest sequence accuracy in non-repetitive regions, while Flye and Raven tend to be the most structurally accurate. In addition, we find that there are unidentified strain-specific characteristics that affect ONT consensus accuracy, despite individual reads having similar levels of accuracy. The differences in consensus accuracy of the ONT reads can preclude accurate assembly regardless of assembly method. These results provide quantitative insight into the best approaches for hybrid assembly of bacterial genomes and the expected levels of structural and sequence accuracy. They also show that there are intrinsic idiosyncratic strain-level differences that inhibit accurate long read bacterial genome assembly. However, we also show it is possible to diagnose problematic assemblies, even in the absence of ground truth, by comparing long-read first and short-read first assemblies.

scholarly journals Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 467
Author(s):  
Aina Ichihara ◽  
Hinako Ojima ◽  
Kazuyoshi Gotoh ◽  
Osamu Matsushita ◽  
Susumu Take ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibody Titer ◽  
Bacterial Genome ◽  
Serum Antibody ◽  
Gene Mutations ◽  
Bacterial Genomes ◽  
Western Blots ◽  
A Genome ◽  
Vaca Gene ◽  
H Pylori

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

scholarly journals Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes

2021 ◽  
Author(s):  
Eric S Tvedte ◽  
Mark Gasser ◽  
Benjamin C Sparklin ◽  
Jane Michalski ◽  
Carl E Hjelmen ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Hybrid Approach ◽  
Cost Effective ◽  
Fruit Fly ◽  
Drosophila Ananassae ◽  
Whole Genome Sequencing Data ◽  
Sequencing Data ◽  
E Coli ◽  
Hybrid Approaches ◽  
Long Read

Abstract The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

scholarly journals Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Matthew R. Olm ◽  
Alexander Crits-Christoph ◽  
Spencer Diamond ◽  
Adi Lavy ◽  
Paula B. Matheus Carnevali ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Ribosomal Proteins ◽  
Large Scale ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Species Discrimination ◽  
Bacterial Genomes ◽  
Discrimination Power

ABSTRACT Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination. IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.

Sign in / Sign up

Export Citation Format

Share Document