scholarly journals Rapid antibiotic resistance predictions from genome sequence data for S. aureus and M. tuberculosis.

2015 ◽  
Author(s):  
Phelim Bradley ◽  
N Claire Gordon ◽  
Timothy M Walker ◽  
Laura Dunn ◽  
Simon Heys ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Single Molecule ◽  
Sequence Data ◽  
Error Rates ◽  
De Bruijn Graph ◽  
Phenotypic Resistance ◽  
Genetic Mechanisms ◽  
Phenotypic Methods ◽  
Sensitivity Specificity ◽  
Scale Control

Rapid and accurate detection of antibiotic resistance in pathogens is an urgent need, affecting both patient care and population-scale control. Microbial genome sequencing promises much, but many barriers exist to its routine deployment. Here, we address these challenges, using a de Bruijn graph comparison of clinical isolate and curated knowledge-base to identify species and predict resistance profile, including minor populations. This is implemented in a package, Mykrobe predictor, for S. aureus and M. tuberculosis, running in under three minutes on a laptop from raw data. For S. aureus, we train and validate in 495/471 samples respectively, finding error rates comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.3%/99.5% across 12 drugs. For M. tuberculosis, we identify species and predict resistance with specificity of 98.5% (training/validating on 1920/1609 samples). Sensitivity of 82.6% is limited by current understanding of genetic mechanisms. We also show that analysis of minor populations increases power to detect phenotypic resistance in second-line drugs without appreciable loss of specificity. Finally, we demonstrate feasibility of an emerging single-molecule sequencing technique.

scholarly journals Multiple Displacement Amplification as a Solution for Low Copy Number Plasmid Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Kuan Yao ◽  
Narjol González-Escalona ◽  
Maria Hoffmann
Keyword(s):  
Antibiotic Resistance ◽  
Single Molecule ◽  
Plasmid Dna ◽  
Copy Number ◽  
Sequence Data ◽  
Multiple Displacement Amplification ◽  
Fast Method ◽  
Alkaline Lysis ◽  
Low Copy Number ◽  
Long Read

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.

scholarly journals Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Phelim Bradley ◽  
N. Claire Gordon ◽  
Timothy M. Walker ◽  
Laura Dunn ◽  
Simon Heys ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mycobacterium Tuberculosis ◽  
Sequence Data ◽  
Error Rates ◽  
Graph Representation ◽  
Clinical Samples ◽  
Resistant Bacteria ◽  
Independent Validation ◽  
Validation Set ◽  
Sensitivity Specificity

Abstract The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package (‘Mykrobe predictor’) that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.

scholarly journals Aligning optical maps to de Bruijn graphs

2019 ◽  
Vol 35 (18) ◽  
pp. 3250-3256 ◽  
Author(s):  
Kingshuk Mukherjee ◽  
Bahar Alipanahi ◽  
Tamer Kahveci ◽  
Leena Salmela ◽  
Christina Boucher
Keyword(s):  
Single Molecule ◽  
Genome Assembly ◽  
Sequence Data ◽  
Supplementary Information ◽  
De Bruijn Graph ◽  
Structural Variations ◽  
Regular Feature ◽  
A Genome ◽  
De Bruijn ◽  
Optical Maps

Abstract Motivation Optical maps are high-resolution restriction maps (Rmaps) that give a unique numeric representation to a genome. Used in concert with sequence reads, they provide a useful tool for genome assembly and for discovering structural variations and rearrangements. Although they have been a regular feature of modern genome assembly projects, optical maps have been mainly used in post-processing step and not in the genome assembly process itself. Several methods have been proposed for pairwise alignment of single molecule optical maps—called Rmaps, or for aligning optical maps to assembled reads. However, the problem of aligning an Rmap to a graph representing the sequence data of the same genome has not been studied before. Such an alignment provides a mapping between two sets of data: optical maps and sequence data which will facilitate the usage of optical maps in the sequence assembly step itself. Results We define the problem of aligning an Rmap to a de Bruijn graph and present the first algorithm for solving this problem which is based on a seed-and-extend approach. We demonstrate that our method is capable of aligning 73% of Rmaps generated from the Escherichia coli genome to the de Bruijn graph constructed from short reads generated from the same genome. We validate the alignments and show that our method achieves an accuracy of 99.6%. We also show that our method scales to larger genomes. In particular, we show that 76% of Rmaps can be aligned to the de Bruijn graph in the case of human data. Availability and implementation The software for aligning optical maps to de Bruijn graph, omGraph is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/omGraph. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Single-molecule sequencing of theDrosophila serratagenome

2016 ◽  
Author(s):  
Scott L. Allen ◽  
Emily K. Delaney ◽  
Artyom Kopp ◽  
Stephen F. Chenoweth
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Sequence Data ◽  
Error Rates ◽  
Model Species ◽  
High Coverage ◽  
Single Molecule Sequencing ◽  
Long Read ◽  
Drosophila Serrata ◽  
High Degree

ABSTRACTLong read sequencing technology promises to greatly enhancede novoassembly of genomes for non-model species. While error rates have been a large stumbling block, sequencing at high coverage allows reads to be self-corrected. Here we sequence andde novoassemble the genome ofDrosophila serrata, a non-model species from themontiumsubgroup that has been well studied for clines and sexual selection. Using 11 PacBio SMRT cells, we generated 12 Gbp of raw sequence data comprising approximately 65x whole genome coverage. Read lengths averaged 8,940 bp (NRead50 12,200) with the longest read at 53 Kbp. We self-corrected reads using the PBDagCon algorithm and assembled the genome using the MHAP algorithm within the PBcR assembler. Total genome length was 198 Mbp with an N50 just under 1 Mbp. Contigs displayed a high degree of arm-level conservation withD. melanogaster. We also provide an initial annotation for this genome usingin silicogene predictions that were supported by RNA-seq data.

scholarly journals Editorial: Evolution of Genetic Mechanisms of Antibiotic Resistance

2019 ◽  
Vol 10 ◽  
Author(s):  
Silvia Buroni ◽  
Simona Pollini ◽  
Gian Maria Rossolini ◽  
Elena Perrin
Keyword(s):  
Antibiotic Resistance ◽  
Genetic Mechanisms

scholarly journals Next Generation Sequencing for the Prediction of the Antibiotic Resistance in Helicobacter pylori: A Literature Review

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 437
Author(s):  
Ilaria Maria Saracino ◽  
Matteo Pavoni ◽  
Angelo Zullo ◽  
Giulia Fiorini ◽  
Tiziana Lazzarotto ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Next Generation Sequencing ◽  
Literature Review ◽  
Molecular Mechanisms ◽  
World Health ◽  
Next Generation ◽  
Culture Methods ◽  
Phenotypic Resistance ◽  
H Pylori ◽  
Generation Sequencing

Background and aims: Only a few antimicrobials are effective against H. pylori, and antibiotic resistance is an increasing problem for eradication therapies. In 2017, the World Health Organization categorized clarithromycin resistant H. pylori as a “high-priority” bacterium. Standard antimicrobial susceptibility testing can be used to prescribe appropriate therapies but is currently recommended only after the second therapeutic failure. H. pylori is, in fact, a “fastidious” microorganism; culture methods are time-consuming and technically challenging. The advent of molecular biology techniques has enabled the identification of molecular mechanisms underlying the observed phenotypic resistance to antibiotics in H. pylori. The aim of this literature review is to summarize the results of original articles published in the last ten years, regarding the use of Next Generation Sequencing, in particular of the whole genome, to predict the antibiotic resistance in H. pylori.Methods: a literature research was made on PubMed. The research was focused on II and III generation sequencing of the whole H. pylori genome. Results: Next Generation Sequencing enabled the detection of novel, rare and complex resistance mechanisms. The prediction of resistance to clarithromycin, levofloxacin and amoxicillin is accurate; for other antimicrobials, such as metronidazole, rifabutin and tetracycline, potential genetic determinants of the resistant status need further investigation.

scholarly journals Preliminary Results on the Prevalence of Salmonella Spp. in Marine Animals Stranded in Sicilian Coasts: Antibiotic Susceptibility Profile and ARGs Detection in the Isolated Strains

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 930
Author(s):  
Delia Gambino ◽  
Sonia Sciortino ◽  
Sergio Migliore ◽  
Lucia Galuppo ◽  
Roberto Puleio ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Susceptibility ◽  
Antibiotic Resistance Genes ◽  
Diffusion Method ◽  
Salmonella Spp ◽  
Marine Animals ◽  
Disk Diffusion Method ◽  
Phenotypic Resistance ◽  
Low Prevalence

The presence of Salmonella spp. in marine animals is a consequence of contamination from terrestrial sources (human activities and animals). Bacteria present in marine environments, including Salmonella spp., can be antibiotic resistant or harbor resistance genes. In this study, Salmonella spp. detection was performed on 176 marine animals stranded in the Sicilian coasts (south Italy). Antibiotic susceptibility, by disk diffusion method and MIC determination, and antibiotic resistance genes, by molecular methods (PCR) of the Salmonella spp. strains, were evaluated. We isolated Salmonella spp. in three animals, though no pathological signs were detected. Our results showed a low prevalence of Salmonella spp. (1.7%) and a low incidence of phenotypic resistance in three Salmonella spp. strains isolated. Indeed, of the three strains, only Salmonella subsp. enterica serovar Typhimurium from S. coeruleoalba and M. mobular showed phenotypic resistance: the first to ampicillin, tetracycline, and sulphamethoxazole, while the latter only to sulphamethoxazole. However, all strains harbored resistance genes (blaTEM, blaOXA, tet(A), tet(D), tet(E), sulI, and sulII). Although the low prevalence of Salmonella spp. found in this study does not represent a relevant health issue, our data contribute to the collection of information on the spread of ARGs, elements involved in antibiotic resistance, now considered a zoonosis in a One Health approach.

scholarly journals Fast and efficient Rmap assembly using the Bi-labelled de Bruijn graph

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Kingshuk Mukherjee ◽  
Massimiliano Rossi ◽  
Leena Salmela ◽  
Christina Boucher
Keyword(s):  
Single Molecule ◽  
De Bruijn Graph ◽  
Anabas Testudineus ◽  
E Coli ◽  
Genome Wide ◽  
A Genome ◽  
De Bruijn ◽  
Optical Maps ◽  
Definition Of ◽  
Numeric Representation

AbstractGenome wide optical maps are high resolution restriction maps that give a unique numeric representation to a genome. They are produced by assembling hundreds of thousands of single molecule optical maps, which are called Rmaps. Unfortunately, there are very few choices for assembling Rmap data. There exists only one publicly-available non-proprietary method for assembly and one proprietary software that is available via an executable. Furthermore, the publicly-available method, by Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006), follows the overlap-layout-consensus (OLC) paradigm, and therefore, is unable to scale for relatively large genomes. The algorithm behind the proprietary method, Bionano Genomics’ Solve, is largely unknown. In this paper, we extend the definition of bi-labels in the paired de Bruijn graph to the context of optical mapping data, and present the first de Bruijn graph based method for Rmap assembly. We implement our approach, which we refer to as rmapper, and compare its performance against the assembler of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) and Solve by Bionano Genomics on data from three genomes: E. coli, human, and climbing perch fish (Anabas Testudineus). Our method was able to successfully run on all three genomes. The method of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) only successfully ran on E. coli. Moreover, on the human genome rmapper was at least 130 times faster than Bionano Solve, used five times less memory and produced the highest genome fraction with zero mis-assemblies. Our software, rmapper is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/Rmapper.

scholarly journals Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions

2018 ◽  
Vol 20 (4) ◽  
pp. 1542-1559 ◽  
Author(s):  
Damla Senol Cali ◽  
Jeremie S Kim ◽  
Saugata Ghose ◽  
Can Alkan ◽  
Onur Mutlu
Keyword(s):  
Sequence Analysis ◽  
Genome Assembly ◽  
Sequence Data ◽  
Error Rates ◽  
Nanopore Sequencing ◽  
Memory Usage ◽  
Sequencing Technology ◽  
Assembly Pipeline ◽  
And Performance ◽  
Polishing Tool

Abstract Nanopore sequencing technology has the potential to render other sequencing technologies obsolete with its ability to generate long reads and provide portability. However, high error rates of the technology pose a challenge while generating accurate genome assemblies. The tools used for nanopore sequence analysis are of critical importance, as they should overcome the high error rates of the technology. Our goal in this work is to comprehensively analyze current publicly available tools for nanopore sequence analysis to understand their advantages, disadvantages and performance bottlenecks. It is important to understand where the current tools do not perform well to develop better tools. To this end, we (1) analyze the multiple steps and the associated tools in the genome assembly pipeline using nanopore sequence data, and (2) provide guidelines for determining the appropriate tools for each step. Based on our analyses, we make four key observations: (1) the choice of the tool for basecalling plays a critical role in overcoming the high error rates of nanopore sequencing technology. (2) Read-to-read overlap finding tools, GraphMap and Minimap, perform similarly in terms of accuracy. However, Minimap has a lower memory usage, and it is faster than GraphMap. (3) There is a trade-off between accuracy and performance when deciding on the appropriate tool for the assembly step. The fast but less accurate assembler Miniasm can be used for quick initial assembly, and further polishing can be applied on top of it to increase the accuracy, which leads to faster overall assembly. (4) The state-of-the-art polishing tool, Racon, generates high-quality consensus sequences while providing a significant speedup over another polishing tool, Nanopolish. We analyze various combinations of different tools and expose the trade-offs between accuracy, performance, memory usage and scalability. We conclude that our observations can guide researchers and practitioners in making conscious and effective choices for each step of the genome assembly pipeline using nanopore sequence data. Also, with the help of bottlenecks we have found, developers can improve the current tools or build new ones that are both accurate and fast, to overcome the high error rates of the nanopore sequencing technology.

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