scholarly journals SMRT-Cappable-seq reveals complex operon variants in bacteria

2018 ◽  
Author(s):  
Bo Yan ◽  
Matthew Boitano ◽  
Tyson Clark ◽  
Laurence Ettwiller
Keyword(s):  
Single Molecule ◽  
Gene Network ◽  
Accurate Identification ◽  
E Coli ◽  
Short Read Sequencing ◽  
Genome Wide ◽  
Long Read ◽  
Definition Of ◽  
Bacterial Genes

AbstractCurrent methods for genome-wide analysis of gene expression requires shredding original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally,in-vivoprocessing of transcripts confounds the accurate identification of the 5’ and 3’ ends of operons. Here we developed a novel methodology called SMRT-Cappable-seq that combines the isolation of unfragmented primary transcripts with single-molecule long read sequencing. Applied toE. coli, this technology results in an unprecedented definition of the transcriptome with 34% of the known operons being extended by at least one gene. Furthermore, 40% of transcription termination sites have read-through that alters the gene content of the operons. As a result, most of the bacterial genes are present in multiple operon variants reminiscent of eukaryotic splicing. By providing an unprecedented granularity in the operon structure, this study represents an important resource for the study of prokaryotic gene network and regulation.

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 Fast and Efficient Rmap Assembly using the Bi-labelled de Bruijn Graph

2021 ◽  
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

Abstract Genome 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 exists very few choices for assembling Rmap data. There exists only one publicly-available non-proprietary method for assembly and one proprietary method that is available via an executable. Furthermore, the publicly-available method, by Valouev et al. (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. (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.(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.

Fatal Respiratory Diphtheria Caused by ß-Lactam–Resistant Corynebacterium diphtheriae

2020 ◽  
Author(s):  
Brian M Forde ◽  
Andrew Henderson ◽  
Elliott G Playford ◽  
David Looke ◽  
Belinda C Henderson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genomic Analysis ◽  
Carbapenem Resistance ◽  
Corynebacterium Diphtheriae ◽  
Penicillin Binding Protein ◽  
Long Read ◽  
Amoxicillin Clavulanic Acid ◽  
Resistance To Penicillin ◽  
High Level

Abstract Background Diphtheria is a potentially fatal respiratory disease caused by toxigenic Corynebacterium diphtheriae. Although resistance to erythromycin has been recognized, β-lactam resistance in toxigenic diphtheria has not been described. Here, we report a case of fatal respiratory diphtheria caused by toxigenic C. diphtheriae resistant to penicillin and all other β-lactam antibiotics, and describe a novel mechanism of inducible carbapenem resistance associated with the acquisition of a mobile resistance element. Methods Long-read whole-genome sequencing was performed using Pacific Biosciences Single Molecule Real-Time sequencing to determine the genome sequence of C. diphtheriae BQ11 and the mechanism of β-lactam resistance. To investigate the phenotypic inducibility of meropenem resistance, short-read sequencing was performed using an Illumina NextSeq500 sequencer on the strain both with and without exposure to meropenem. Results BQ11 demonstrated high-level resistance to penicillin (benzylpenicillin minimum inhibitory concentration [MIC] ≥ 256 μg/ml), β-lactam/β-lactamase inhibitors and cephalosporins (amoxicillin/clavulanic acid MIC ≥ 256 μg/mL; ceftriaxone MIC ≥ 8 μg/L). Genomic analysis of BQ11 identified acquisition of a novel transposon carrying the penicillin-binding protein (PBP) Pbp2c, responsible for resistance to penicillin and cephalosporins. When strain BQ11 was exposed to meropenem, selective pressure drove amplification of the transposon in a tandem array and led to a corresponding change from a low-level to a high-level meropenem-resistant phenotype. Conclusions We have identified a novel mechanism of inducible antibiotic resistance whereby isolates that appear to be carbapenem susceptible on initial testing can develop in vivo resistance to carbapenems with repeated exposure. This phenomenon could have significant implications for the treatment of C. diphtheriae infection, and may lead to clinical failure.

scholarly journals Elevated virulence of an emerging viral genotype as a driver of honeybee loss

2016 ◽  
Vol 283 (1833) ◽  
pp. 20160811 ◽  
Author(s):  
Dino P. McMahon ◽  
Myrsini E. Natsopoulou ◽  
Vincent Doublet ◽  
Matthias Fürst ◽  
Silvio Weging ◽  
...  
Keyword(s):  
Emerging Infectious Diseases ◽  
Genetic Identity ◽  
Accurate Identification ◽  
Deformed Wing Virus ◽  
Species Decline ◽  
Biodiversity Crisis ◽  
Pathogen Virulence ◽  
Population Loss ◽  
Genome Wide

Emerging infectious diseases (EIDs) have contributed significantly to the current biodiversity crisis, leading to widespread epidemics and population loss. Owing to genetic variation in pathogen virulence, a complete understanding of species decline requires the accurate identification and characterization of EIDs. We explore this issue in the Western honeybee, where increasing mortality of populations in the Northern Hemisphere has caused major concern. Specifically, we investigate the importance of genetic identity of the main suspect in mortality, deformed wing virus (DWV), in driving honeybee loss. Using laboratory experiments and a systematic field survey, we demonstrate that an emerging DWV genotype (DWV-B) is more virulent than the established DWV genotype (DWV-A) and is widespread in the landscape. Furthermore, we show in a simple model that colonies infected with DWV-B collapse sooner than colonies infected with DWV-A. We also identify potential for rapid DWV evolution by revealing extensive genome-wide recombination in vivo . The emergence of DWV-B in naive honeybee populations, including via recombination with DWV-A, could be of significant ecological and economic importance. Our findings emphasize that knowledge of pathogen genetic identity and diversity is critical to understanding drivers of species decline.

scholarly journals Long-range single-molecule mapping of chromatin modification in eukaryotes

2021 ◽  
Author(s):  
Zhe Weng ◽  
Fengying Ruan ◽  
Weitian Chen ◽  
Zhe Xie ◽  
Yeming Xie ◽  
...  
Keyword(s):  
Histone Modification ◽  
Long Range ◽  
Single Molecule ◽  
Protein A ◽  
Chromatin Modification ◽  
Protein Binding Sites ◽  
Short Read Sequencing ◽  
Third Generation Sequencing ◽  
Long Read ◽  
Generation Sequencing

The epigenetic modifications of histones are essential marks related to the development and disease pathogenesis, including human cancers. Mapping histone modification has emerged as the widely used tool for studying epigenetic regulation. However, existing approaches limited by fragmentation and short-read sequencing cannot provide information about the long-range chromatin states and represent the average chromatin status in samples. We leveraged the advantage of long read sequencing to develop a method "BIND&MODIFY" for profiling the histone modification of individual DNA fiber. Our approach is based on the recombinant fused protein A-EcoGII, which tethers the methyltransferase EcoGII to the protein binding sites and locally labels the neighboring DNA regions through artificial methylations. We demonstrate that the aggregated BIND&MODIFY signal matches the bulk-level ChIP-seq and CUT&TAG, observe the single-molecule heterogenous histone modification status, and quantify the correlation between distal elements. This method could be an essential tool in the future third-generation sequencing ages.

scholarly journals DNA sliding and loop formation by E. coli SMC complex: MukBEF

2021 ◽  
Author(s):  
man zhou
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Interaction ◽  
Loop Formation ◽  
Unknown Mechanism ◽  
E Coli ◽  
Dna Compaction ◽  
And Function ◽  
Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

scholarly journals Mapping and phasing of structural variation in patient genomes using nanopore sequencing

2017 ◽  
Author(s):  
Mircea Cretu Stancu ◽  
Markus J. van Roosmalen ◽  
Ivo Renkens ◽  
Marleen Nieboer ◽  
Sjors Middelkamp ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Human Genetic Disease ◽  
Structural Genomic ◽  
Short Read ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Long Read ◽  
Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

scholarly journals Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

2020 ◽  
Vol 48 (15) ◽  
pp. 8490-8508 ◽  
Author(s):  
Sarah S Henrikus ◽  
Camille Henry ◽  
Amy E McGrath ◽  
Slobodan Jergic ◽  
John P McDonald ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Pol Iv ◽  
Dna Polymerase Iv

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

scholarly journals Kohdista: an efficient method to index and query possible Rmap alignments

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Martin D. Muggli ◽  
Simon J. Puglisi ◽  
Christina Boucher
Keyword(s):  
Single Molecule ◽  
Restriction Enzymes ◽  
Consensus Approach ◽  
E Coli ◽  
Genome Wide ◽  
Alignment Problem ◽  
Optical Map ◽  
Optical Maps ◽  
Map Data ◽  
Genomic Regions

Abstract Background Genome-wide optical maps are ordered high-resolution restriction maps that give the position of occurrence of restriction cut sites corresponding to one or more restriction enzymes. These genome-wide optical maps are assembled using an overlap-layout-consensus approach using raw optical map data, which are referred to as Rmaps. Due to the high error-rate of Rmap data, finding the overlap between Rmaps remains challenging. Results We present Kohdista, which is an index-based algorithm for finding pairwise alignments between single molecule maps (Rmaps). The novelty of our approach is the formulation of the alignment problem as automaton path matching, and the application of modern index-based data structures. In particular, we combine the use of the Generalized Compressed Suffix Array (GCSA) index with the wavelet tree in order to build Kohdista. We validate Kohdista on simulated E. coli data, showing the approach successfully finds alignments between Rmaps simulated from overlapping genomic regions. Conclusion we demonstrate Kohdista is the only method that is capable of finding a significant number of high quality pairwise Rmap alignments for large eukaryote organisms in reasonable time.

scholarly journals Genome-wide comparison of phage M13-infected vs. uninfectedEscherichia coli

2005 ◽  
Vol 51 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Fredrik Karlsson ◽  
Ann-Christin Malmborg-Hager ◽  
Ann-Sofie Albrekt ◽  
Carl A.K Borrebaeck
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Acid Stress ◽  
Phage Infection ◽  
Transcriptional Analysis ◽  
Filamentous Phage ◽  
Phosphotransferase System ◽  
E Coli ◽  
Genome Wide

To identify Escherichia coli genes potentially regulated by filamentous phage infection, we used oligonucleotide microarrays. Genome-wide comparison of phage M13-infected and uninfected E. coli, 2 and 20 min after infection, was performed. The analysis revealed altered transcription levels of 12 E. coli genes in response to phage infection, and the observed regulation of phage genes correlated with the known in vivo pattern of M13 mRNA species. Ten of the 12 host genes affected could be grouped into 3 different categories based on cellular function, suggesting a coordinated response. The significantly upregulated genes encode proteins involved in reactions of the energy-generating phosphotransferase system and transcription processing, which could be related to phage transcription. No genes belonging to any known E. coli stress response pathways were scored as upregulated. Furthermore, phage infection led to significant downregulation of transcripts of the bacterial genes gadA, gadB, hdeA, gadE, slp, and crl. These downregulated genes are normally part of the host stress response mechanisms that protect the bacterium during conditions of acid stress and stationary phase transition. The phage-infected cells demonstrated impaired function of the oxidative and the glutamate-dependent acid resistance systems. Thus, global transcriptional analysis and functional analysis revealed previously unknown host responses to filamentous phage infection.Key words: filamentous phage infection, global transcriptional analysis, AR, Escherichia coli.

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