scholarly journals Genomic Surveillance for Antimicrobial Resistance inMannheimia haemolyticaUsing Nanopore Single Molecule Sequencing Technology

2018 ◽  
Author(s):  
Alexander Lim ◽  
Bryan Naidenov ◽  
Haley Bates ◽  
Karyn Willyerd ◽  
Timothy Snider ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Single Molecule ◽  
Resistant Strain ◽  
De Novo ◽  
Gene Annotation ◽  
Cost Effective ◽  
Multidrug Resistant ◽  
Oxford Nanopore ◽  
Long Read

AbstractDisruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain ofMannheimia haemolytica, isolated from pneumonic bovine lung samples, were respectively sequenced at 146x and 111x coverage with Oxford Nanopore Technologies MinION.De novoassembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including beta lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Antibiotic resistance phenotypes of theM. haemolyticastrains were confirmed with minimum inhibitory concentration (MIC) assays. The sequencing capacity of highly portable MinION devices was verified by sub-sampling sequencing reads; potential for antimicrobial resistance determined by identification of resistance genes in the draft assemblies with as little as 5,437 MinION reads corresponded to all classes of MIC assays. The resulting quality assemblies and AMR gene annotation highlight efficiency of ultra long-read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.

scholarly journals Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

2019 ◽  
Author(s):  
Mitchell R. Vollger ◽  
Glennis A. Logsdon ◽  
Peter A. Audano ◽  
Arvis Sulovari ◽  
David Porubsky ◽  
...  
Keyword(s):  
Human Genome ◽  
Single Molecule ◽  
Tandem Repeats ◽  
De Novo ◽  
Sequence Data ◽  
Gene Annotation ◽  
Hydatidiform Mole ◽  
High Fidelity ◽  
Human Genomes ◽  
Long Read

AbstractThe sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective stand-alone technology for de novo assembly of human genomes.

scholarly journals Ventilator-Associated Pneumonia: The Role of Emerging Diagnostic Technologies

2017 ◽  
Vol 38 (03) ◽  
pp. 253-263 ◽  
Author(s):  
Carey-Ann Burnham ◽  
Marin Kollef
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Cost Effective ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Ventilator Associated Pneumonia ◽  
Rapid Diagnostics ◽  
Acinetobacter Species ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Diagnostic Technologies

AbstractAntibiotic resistance has emerged as a key determinant of outcome in patients with serious infections along with the virulence of the underlying pathogen. Within the intensive care unit (ICU) setting, ventilator-associated pneumonia (VAP) is a common nosocomial infection that is frequently caused by multidrug-resistant bacteria. Antimicrobial resistance is a growing challenge in the care of critically ill patients. Escalating rates of antibiotic resistance add substantially to the morbidity, mortality, and cost related to infection in the ICU. Both gram-positive organisms, such as methicillin-resistant Staphylococcus aureus and vancomycin-intermediate S. aureus, and gram-negative bacteria, including Pseudomonas aeruginosa, Acinetobacter species, carbapenem-resistant Enterobacteriaceae, such as the Klebsiella pneumoniae carbapenemase–producing bacteria, and extended spectrum β-lactamase organisms, have contributed to the escalating rates of resistance seen in VAP and other nosocomial infections. The rising rates of antimicrobial resistance have led to the routine empiric administration of broad-spectrum antibiotics even when bacterial infection is not documented. Moreover, there are several new broader-spectrum antibiotics that have recently become available and others scheduled for approval in the near future. The challenge to ICU clinicians is how to most effectively utilize these agents to maximize patient benefits while minimizing further emergence of resistance. Use of rapid diagnostics may hold the key for achieving this important balance. There is an urgent need for integrating the administration of new and existing antibiotics with the emerging rapid diagnostic technologies in a way that is both cost-effective and sustainable for the long run.

scholarly journals lra: A long read aligner for sequences and contigs

2021 ◽  
Vol 17 (6) ◽  
pp. e1009078
Author(s):  
Jingwen Ren ◽  
Mark J. P. Chaisson
Keyword(s):  
Dynamic Programming ◽  
Single Molecule ◽  
De Novo Assembly ◽  
De Novo ◽  
Concave Function ◽  
Single Molecule Sequencing ◽  
Link Type ◽  
Oxford Nanopore ◽  
Concave Cost ◽  
Long Read

It is computationally challenging to detect variation by aligning single-molecule sequencing (SMS) reads, or contigs from SMS assemblies. One approach to efficiently align SMS reads is sparse dynamic programming (SDP), where optimal chains of exact matches are found between the sequence and the genome. While straightforward implementations of SDP penalize gaps with a cost that is a linear function of gap length, biological variation is more accurately represented when gap cost is a concave function of gap length. We have developed a method, lra, that uses SDP with a concave-cost gap penalty, and used lra to align long-read sequences from PacBio and Oxford Nanopore (ONT) instruments as well as de novo assembly contigs. This alignment approach increases sensitivity and specificity for SV discovery, particularly for variants above 1kb and when discovering variation from ONT reads, while having runtime that are comparable (1.05-3.76×) to current methods. When applied to calling variation from de novo assembly contigs, there is a 3.2% increase in Truvari F1 score compared to minimap2+htsbox. lra is available in bioconda (https://anaconda.org/bioconda/lra) and github (https://github.com/ChaissonLab/LRA).

scholarly journals Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation

2016 ◽  
Author(s):  
Sergey Koren ◽  
Brian P. Walenz ◽  
Konstantin Berlin ◽  
Jason R. Miller ◽  
Nicholas H. Bergman ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Error Rates ◽  
Celera Assembler ◽  
Oxford Nanopore ◽  
Long Read ◽  
Reference Quality ◽  
Order Of Magnitude ◽  
Assembly Algorithms ◽  
Oxford Nanopore Technologies

AbstractLong-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.

scholarly journals Comparison of long-read methods for sequencing and assembly of a plant genome

GigaScience ◽  
2020 ◽  
Vol 9 (12) ◽  
Author(s):  
Valentine Murigneux ◽  
Subash Kumar Rai ◽  
Agnelo Furtado ◽  
Timothy J C Bruxner ◽  
Wei Tian ◽  
...  
Keyword(s):  
De Novo ◽  
Cost Effective ◽  
Genome Project ◽  
Plant Genome ◽  
Sequencing Data ◽  
Pacific Biosciences ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
The Cost

Abstract Background Sequencing technologies have advanced to the point where it is possible to generate high-accuracy, haplotype-resolved, chromosome-scale assemblies. Several long-read sequencing technologies are available, and a growing number of algorithms have been developed to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology, as well as the most appropriate software for assembly and polishing. It is thus important to benchmark different approaches applied to the same sample. Results Here, we report a comparison of 3 long-read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION), and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements. Conclusions The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput, and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

scholarly journals lra: the Long Read Aligner for Sequences and Contigs

2020 ◽  
Author(s):  
Jingwen Ren ◽  
Mark JP Chaisson
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Data Types ◽  
Single Molecule Sequencing ◽  
Detection Algorithms ◽  
Link Type ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Linear Cost

AbstractMotivationIt is computationally challenging to detect variation by aligning long reads from single-molecule sequencing (SMS) instruments, or megabase-scale contigs from SMS assemblies. One approach to efficiently align long sequences is sparse dynamic programming (SDP), where exact matches are found between the sequence and the genome, and optimal chains of matches are found representing a rough alignment. Sequence variation is more accurately modeled when alignments are scored with a gap penalty that is a convex function of the gap length. Because previous implementations of SDP used a linear-cost gap function that does not accurately model variation, and implementations of alignment that have a convex gap penalty are either inefficient or use heuristics, we developed a method, lra, that uses SDP with a convex-cost gap penalty. We use lra to align long-read sequences from PacBio and Oxford Nanopore (ONT) instruments as well as de novo assembly contigs.ResultsAcross all data types, the runtime of lra is between 52-168% of the state of the art aligner minimap2 when generating SAM alignment, and 9-15% of an alternative method, ngmlr. This alignment approach may be used to provide additional evidence of SV calls in PacBio datasets, and an increase in sensitivity and specificity on ONT data with current SV detection algorithms. The number of calls discovered using pbsv with lra alignments are within 98.3-98.6% of calls made from minimap2 alignments on the same data, and give a nominal 0.2-0.4% increase in F1 score by Truvari analysis. On ONT data with SV called using Sniffles, the number of calls made from lra alignments is 3% greater than minimap2-based calls, and 30% greater than ngmlr based calls, with a 4.6-5.5% increase in Truvari F1 score. When applied to calling variation from de novo assembly contigs, there is a 5.8% increase in SV calls compared to minimap2+paftools, with a 4.3% increase in Truvari F1 score.Availability and implementationAvailable in bioconda: https://anaconda.org/bioconda/lra and github: https://github.com/ChaissonLab/[email protected], [email protected]

scholarly journals The power of single molecule real-time sequencing technology in the de novo assembly of a eukaryotic genome

2015 ◽  
Author(s):  
Hiroaki Sakai ◽  
Ken Naito ◽  
Eri Ogiso-Tanaka ◽  
Yu Takahashi ◽  
Kohtaro Iseki ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
De Novo ◽  
Gene Annotation ◽  
Cost Effective ◽  
Detailed Comparison ◽  
Azuki Bean ◽  
Eukaryotic Genome ◽  
Model Organisms ◽  
Sequencing Technology

Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms. However, a large portion of the genomes still remains unassembled. We reconstructed azuki bean (Vigna angularis) genome using single molecule real-time (SMRT) sequencing technology and achieved the best contiguity and coverage among currently assembled legume crops. The SMRT-based assembly produced 100 times longer contigs with 100 times smaller amount of gaps compared to the SGS-based assemblies. A detailed comparison between the assemblies revealed that the SMRT-based assembly enabled a more comprehensive gene annotation than the SGS-based assemblies where thousands of genes were missing or fragmented. A chromosome-scale assembly was generated based on the high-density genetic map, covering 86% of the azuki bean genome. We demonstrated that SMRT technology, though still needed to be assisted by SGS data, can achieve a near-complete assembly of a eukaryotic genome.

scholarly journals Comparison of long read methods for sequencing and assembly of a plant genome

2020 ◽  
Author(s):  
Valentine Murigneux ◽  
Subash Kumar Rai ◽  
Agnelo Furtado ◽  
Timothy J.C. Bruxner ◽  
Wei Tian ◽  
...  
Keyword(s):  
De Novo ◽  
Cost Effective ◽  
Genome Project ◽  
Plant Genome ◽  
Sequencing Data ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
The Cost ◽  
Genome Assemblies

AbstractSequencing technologies have advanced to the point where it is possible to generate high accuracy, haplotype resolved, chromosome scale assemblies. Several long read sequencing technologies are available on the market and a growing number of algorithms have been developed over the last years to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology as well as the most appropriate software for assembly and polishing. For this reason, it is important to benchmark different approaches applied to the same sample. Here, we report a comparison of three long read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION) and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of PacBio and Nanopore reads. Results obtained from combining long read technologies or short read and long read technologies are also presented. The assemblies were compared for contiguity, accuracy and completeness as well as sequencing costs and DNA material requirements. Overall, the three long read technologies produced highly contiguous and complete genome assemblies of Macadamia jansenii. At the time of sequencing, the cost associated with each method was significantly different but continuous improvements in technologies have resulted in greater accuracy, increased throughput and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

scholarly journals Single-molecule sequencing of long DNA molecules allows high contiguity de novo genome assembly for the fungus fly, Sciara coprophila

2020 ◽  
Author(s):  
John M. Urban ◽  
Michael S. Foulk ◽  
Jacob E. Bliss ◽  
C. Michelle Coleman ◽  
Nanyan Lu ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Single Molecule ◽  
De Novo ◽  
Draft Genome ◽  
Chromosome Elimination ◽  
Future Research ◽  
Paternal Chromosome ◽  
Oxford Nanopore ◽  
Dna Modifications ◽  
Long Read

ABSTRACTThe lower Dipteran fungus fly, Sciara coprophila, has many unique biological features. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in aiding these studies. We approached assembling the Sciara genome using multiple sequencing technologies: PacBio, Oxford Nanopore MinION, and Illumina. To find an optimal assembly using these datasets, we generated 44 Illumina assemblies using 7 short-read assemblers and 50 long-read assemblies of PacBio and MinION sequence data using 6 long-read assemblers. We ranked assemblies using a battery of reference-free metrics, and scaffolded a subset of the highest-ranking assemblies using BioNano Genomics optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. Moreover, we anchored nearly half of the Sciara genome sequence into chromosomes. Finally, we used the signal level of both the PacBio and Oxford Nanopore data to explore the presence or absence of DNA modifications in the Sciara genome since DNA modifications may play a role in imprinting in Sciara, as they do in mammals. These data serve as the foundation for future research by the growing community studying the unique features of this emerging model system.

Antimicrobial Resistance and Presence of Class 1 Integrons Among Different Serotypes of Salmonella spp. Recovered From Children with Diarrhea in Tehran, Iran

2020 ◽  
Vol 20 (2) ◽  
pp. 160-166
Author(s):  
Seyedeh Hanieh Eshaghi Zadeh ◽  
Hossein Fahimi ◽  
Fatemeh Fardsanei ◽  
Mohammad Mehdi Soltan Dallal
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Multidrug Resistant ◽  
Salmonella Spp ◽  
Class 1 Integron ◽  
Class 1 Integrons ◽  
Food Borne ◽  
Class 1 ◽  
Resistance Patterns ◽  
C 30

Background: Salmonellosis is a major food-borne disease worldwide. The increasing prevalence of antimicrobial resistance among food-borne pathogens such as Salmonella spp. is concerning. Objective: The main objective of this study is to identify class 1 integron genes and to determine antibiotic resistance patterns among Salmonella isolates from children with diarrhea. Methods: A total of 30 Salmonella isolates were recovered from children with diarrhea. The isolates were characterized for antimicrobial susceptibility and screened for the presence of class 1 integron genes (i.e. intI1, sulI1, and qacEΔ1). Results: The most prevalent serotype was Enteritidis 36.7%, followed by Paratyphi C (30%), and Typhimurium (16.7%). The highest rates of antibiotic resistance were obtained for nalidixic acid (53.3%), followed by streptomycin (40%), and tetracycline (36.7%). Regarding class 1 integrons, 36.7%, 26.7%, and 33.3% of the isolates carried intI1, SulI, and qacEΔ1, respectively, most of which (81.8%) were multidrug-resistant (MDR). Statistical analysis revealed that the presence of class 1 integron was significantly associated with resistance to streptomycin and tetracycline (p = 0.042). However, there was no association between class 1 integron and other antibiotics used in this study (p > 0.05). Conclusion: The high frequency of integron class 1 gene in MDR Salmonella strains indicates that these mobile genetic elements are versatile among different Salmonella serotypes, and associated with reduced susceptibility to many antimicrobials.

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