scholarly journals Methylation analysis of Klebsiella pneumoniae from Portuguese hospitals

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anton Spadar ◽  
João Perdigão ◽  
Jody Phelan ◽  
James Charleston ◽  
Ana Modesto ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Infectious Agent ◽  
Sequencing Platform ◽  
Extended Spectrum Beta Lactamase ◽  
Sequencing Technologies ◽  
Dna Adenine Methylase ◽  
Long Read ◽  
Foreign Dna ◽  
Reference Genomes ◽  
Restriction Modification

AbstractKlebsiella pneumoniae is an important nosocomial infectious agent with a high antimicrobial resistance (AMR) burden. The application of long read sequencing technologies is providing insights into bacterial chromosomal and putative extra-chromosomal genetic elements (PEGEs) associated with AMR, but also epigenetic DNA methylation, which is thought to play a role in cleavage of foreign DNA and expression regulation. Here, we apply the PacBio sequencing platform to eight Portuguese hospital isolates, including one carbapenemase producing isolate, to identify methylation motifs. The resulting assembled chromosomes were between 5.2 and 5.5Mbp in length, and twenty-six PEGEs were found. Four of our eight samples carry blaCTX-M-15, a dominant Extended Spectrum Beta Lactamase in Europe. We identified methylation motifs that control Restriction–Modification systems, including GATC of the DNA adenine methylase (Dam), which methylates N6-methyladenine (m6A) across all our K. pneumoniae assemblies. There was a consistent lack of methylation by Dam of the GATC motif downstream of two genes: fosA, a locus associated with low level fosfomycin resistance, and tnpB transposase on IncFIB(K) plasmids. Overall, we have constructed eight high quality reference genomes of K. pneumoniae, with insights into horizontal gene transfer and methylation m6A motifs.

scholarly journals Improving the Chromosome-Level Genome Assembly of the Siamese Fighting Fish (Betta splendens) in a University Master’s Course

2020 ◽  
Vol 10 (7) ◽  
pp. 2179-2183 ◽  
Author(s):  
Stefan Prost ◽  
Malte Petersen ◽  
Martin Grethlein ◽  
Sarah Joy Hahn ◽  
Nina Kuschik-Maczollek ◽  
...  
Keyword(s):  
Genome Assembly ◽  
High Throughput Sequencing ◽  
Siamese Fighting Fish ◽  
Betta Splendens ◽  
High Quality ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Chromosome Level

Ever decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university master’s course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behavior. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published Hi-C data. The use of ∼35x nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using the Hi-C data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 96.1% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly. We present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university master’s course. The use of ∼35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

scholarly journals Long read sequencing reveals Poxvirus evolution through rapid homogenization of gene arrays

2018 ◽  
Author(s):  
Thomas A. Sasani ◽  
Kelsey R. Cone ◽  
Aaron R. Quinlan ◽  
Nels C. Elde
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Single Nucleotide Variants ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Gene Copy Number Variation ◽  
Large Gene ◽  
Long Read ◽  
Number Variation

AbstractLarge DNA viruses rapidly evolve to defeat host defenses. Poxvirus adaptation can involve combinations of recombination-driven gene copy number variation and beneficial single nucleotide variants (SNVs) at the same locus, yet how these distinct mechanisms of genetic diversification might simultaneously facilitate adaptation to immune blocks is unknown. We performed experimental evolution with a vaccinia virus population harboring a SNV in a gene actively undergoing copy number amplification. Comparisons of virus genomes using the Oxford Nanopore Technologies sequencing platform allowed us to phase SNVs within large gene copy arrays for the first time, and uncovered a mechanism of adaptive SNV homogenization reminiscent of gene conversion, which is actively driven by selection. Our work reveals a new mechanism for the fluid gain of beneficial mutations in genetic regions undergoing active recombination in viruses, and illustrates the value of long read sequencing technologies for investigating complex genome dynamics in diverse biological systems.

scholarly journals Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

2022 ◽  
Vol 19 (1) ◽  
Author(s):  
Ádám Fülöp ◽  
Gábor Torma ◽  
Norbert Moldován ◽  
Kálmán Szenthe ◽  
Ferenc Bánáti ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Open Reading Frames ◽  
Integrative Approach ◽  
Splice Isoforms ◽  
Sequencing Platform ◽  
Barr Virus ◽  
Pacific Biosciences ◽  
Sequencing Technologies ◽  
Long Read ◽  
Epstein Barr

Abstract Background Epstein–Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read-sequencing and Pacific Biosciences RS II-based long-read sequencing technologies. Since the various sequencing methods have distinct strengths and limitations, the use of multiplatform approaches have proven to be valuable. The aim of this study is to provide a more complete picture on the transcriptomic architecture of EBV. Methods In this work, we apply the Oxford Nanopore Technologies MinION (long-read sequencing) platform for the generation of novel transcriptomic data, and integrate these with other’s data generated by another LRS approach, Pacific BioSciences RSII sequencing and Illumina CAGE-Seq and Poly(A)-Seq approaches. Both amplified and non-amplified cDNA sequencings were applied for the generation of sequencing reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. Results This study detected novel genes embedded into longer host genes containing 5′-truncated in-frame open reading frames, which potentially encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel replication-origin-associated transcripts. Additionally, novel mono- and multigenic transcripts were identified. An intricate meshwork of transcriptional overlaps was revealed. Conclusions An integrative approach applying multi-technique sequencing technologies is suitable for reliable identification of complex transcriptomes because each techniques has different advantages and limitations, and the they can be used for the validation of the results obtained by a particular approach.

scholarly journals Towards complete and error-free genome assemblies of all vertebrate species

Nature ◽  
2021 ◽  
Vol 592 (7856) ◽  
pp. 737-746 ◽  
Author(s):  
Arang Rhie ◽  
Shane A. McCarthy ◽  
Olivier Fedrigo ◽  
Joana Damas ◽  
Giulio Formenti ◽  
...  
Keyword(s):  
Cost Effective ◽  
Lessons Learned ◽  
Vertebrate Species ◽  
High Quality ◽  
Protein Coding ◽  
Sequencing Technologies ◽  
Long Read ◽  
Genome Assemblies ◽  
Assembly Error ◽  
Reference Genomes

AbstractHigh-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1–4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

scholarly journals Improving the chromosome-level genome assembly of the Siamese fighting fish (Betta splendens) in a university Master’s course

2020 ◽  
Author(s):  
Stefan Prost ◽  
Malte Petersen ◽  
Martin Grethlein ◽  
Sarah Joy Hahn ◽  
Nina Kuschik-Maczollek ◽  
...  
Keyword(s):  
Genome Assembly ◽  
High Throughput Sequencing ◽  
Siamese Fighting Fish ◽  
Betta Splendens ◽  
High Quality ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Chromosome Level

AbstractBackgroundEver decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university Master’s course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behaviour. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published HiC data.FindingsThe use of nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using previously published HiC data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 95.8% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly.ConclusionWe present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university Master’s course. The use of ~35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

scholarly journals Comparative Analysis of PacBio and Oxford Nanopore Sequencing Technologies for Transcriptomic Landscape Identification of Penaeus monodon

Life ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 862
Author(s):  
Zulema Udaondo ◽  
Kanchana Sittikankaew ◽  
Tanaporn Uengwetwanit ◽  
Thidathip Wongsurawat ◽  
Chutima Sonthirod ◽  
...  
Keyword(s):  
Penaeus Monodon ◽  
Influential Factors ◽  
Read Length ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Research Fields ◽  
Oxford Nanopore ◽  
Long Read ◽  
Assembly Features ◽  
Sequencing Platforms

With the advantages that long-read sequencing platforms such as Pacific Biosciences (Menlo Park, CA, USA) (PacBio) and Oxford Nanopore Technologies (Oxford, UK) (ONT) can offer, various research fields such as genomics and transcriptomics can exploit their benefits. Selecting an appropriate sequencing platform is undoubtedly crucial for the success of the research outcome, thus there is a need to compare these long-read sequencing platforms and evaluate them for specific research questions. This study aims to compare the performance of PacBio and ONT platforms for transcriptomic analysis by utilizing transcriptome data from three different tissues (hepatopancreas, intestine, and gonads) of the juvenile black tiger shrimp, Penaeus monodon. We compared three important features: (i) main characteristics of the sequencing libraries and their alignment with the reference genome, (ii) transcript assembly features and isoform identification, and (iii) correlation of the quantification of gene expression levels for both platforms. Our analyses suggest that read-length bias and differences in sequencing throughput are highly influential factors when using long reads in transcriptome studies. These comparisons can provide a guideline when designing a transcriptome study utilizing these two long-read sequencing technologies.

scholarly journals 626. Mobile Genetic Element Dynamics of Co-Circulating Klebsiella pneumoniae Sequence Types Carrying blaKPC in Houston, Texas

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S290-S291
Author(s):  
William C Shropshire ◽  
An Q Dinh ◽  
William R Miller ◽  
Heather Ecklund ◽  
Audrey Wanger ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Carbapenem Resistance ◽  
Resistance Mechanisms ◽  
Metropolitan Region ◽  
Genetic Lineage ◽  
Genomic Context ◽  
Hospital System ◽  
Sequencing Technologies ◽  
Long Read ◽  
Metropolitan Hospital

Abstract Background Carbapenem-resistant Klebsiella pneumoniae (CR-Kpn) are a significant cause of hospital-associated infections. Class A β-lactamases, e.g., Klebsiella pneumoniae carbapenemases (KPCs), are major contributors to carbapenem resistance. Sequence type 258 (ST258) is the most common genetic lineage of CR-Kpn associated with blaKPC carriage. Recently, a newly emergent lineage ST307 has been identified within the Houston metropolitan region. The transmission of blaKPC and other antimicrobial resistance (AMR) genes is driven largely by exchange of mobile genetic elements (MGEs). We sought to describe the dynamics of horizontal gene transfer (HGT) in particular between co-circulating strains of ST307 and ST258. Methods Long-read sequencing technologies allow us to resolve plasmid sequences and their associated AMR genes as well as characterize a comprehensive range of MGEs enabling transmission of these clinically important resistance mechanisms. CR-Kpn isolates were collected as part of a study to describe CRE burden within a Houston metropolitan hospital system. The Oxford Nanopore Technology (ONT) GridION X5 was used for long-read sequencing with Illumina short-read data used to refine and generate high-quality, consensus assemblies. A custom bioinformatic pipeline was used to resolve plasmid structures and identify the genomic context of plasmids carrying blaKPC variants. Results 95 Kpn isolates were collected from May to December 2017. Phylogenetic and in silico MLST analysis revealed 38/95 (40%) and 35/95 (37%) were ST258 and ST307, respectively. 86% of Kpn isolates carried one or more IncF-type conjugative plasmids, which were the prime vectors for blaKPC intercellular transmission. Interestingly, we found similar AMR-harboring plasmids within ST258 and ST307 composed of mosaic, modular IS26 and Tn3-like transposase mediated elements that carried multiple AMR determinants including blaKPC variants (Figure 1). Conclusion We were able to characterize mechanisms by which ST307 and ST258 lineages may transfer AMR determinants. There are clinically relevant implications to these HGT events that occur between these lineages as they may provide insights into how resistance differentially develops. Disclosures All authors: No reported disclosures.

scholarly journals Towards complete and error-free genome assemblies of all vertebrate species

2020 ◽  
Author(s):  
Arang Rhie ◽  
Shane A. McCarthy ◽  
Olivier Fedrigo ◽  
Joana Damas ◽  
Giulio Formenti ◽  
...  
Keyword(s):  
Cost Effective ◽  
Lessons Learned ◽  
Vertebrate Species ◽  
High Quality ◽  
New Era ◽  
Sequencing Technologies ◽  
Long Read ◽  
Cartilaginous Fishes ◽  
Genome Assemblies ◽  
Reference Genomes

AbstractHigh-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are only available for a few non-microbial species1–4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling the most accurate and complete reference genomes to date. Here we summarize these developments, introduce a set of quality standards, and present lessons learned from sequencing and assembling 16 species representing major vertebrate lineages (mammals, birds, reptiles, amphibians, teleost fishes and cartilaginous fishes). We confirm that long-read sequencing technologies are essential for maximizing genome quality and that unresolved complex repeats and haplotype heterozygosity are major sources of error in assemblies. Our new assemblies identify and correct substantial errors in some of the best historical reference genomes. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an effort to generate high-quality, complete reference genomes for all ~70,000 extant vertebrate species and help enable a new era of discovery across the life sciences.

scholarly journals MiniScrub: de novo long read scrubbing using approximate alignment and deep learning

2018 ◽  
Author(s):  
Nathan LaPierre ◽  
Rob Egan ◽  
Wei Wang ◽  
Zhong Wang
Keyword(s):  
Open Source Software ◽  
Genome Assembly ◽  
Large Scale ◽  
Structural Variation ◽  
De Novo ◽  
Error Rates ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Reference Genomes

AbstractLong read sequencing technologies such as Oxford Nanopore can greatly de-crease the complexity of de novo genome assembly and large structural variation iden-tification. Currently Nanopore reads have high error rates, and the errors often cluster into low-quality segments within the reads. Many methods for resolving these errors require access to reference genomes, high-fidelity short reads, or reference genomes, which are often not available. De novo error correction modules are available, often as part of assembly tools, but large-scale errors still remain in resulting assemblies, motivating further innovation in this area. We developed a novel Convolutional Neu-ral Network (CNN) based method, called MiniScrub, for de novo identification and subsequent “scrubbing” (removal) of low-quality Nanopore read segments. MiniScrub first generates read-to-read alignments by MiniMap, then encodes the alignments into images, and finally builds CNN models to predict low-quality segments that could be scrubbed based on a customized quality cutoff. Applying MiniScrub to real world con-trol datasets under several different parameters, we show that it robustly improves read quality. Compared to raw reads, de novo genome assembly with scrubbed reads pro-duces many fewer mis-assemblies and large indel errors. We propose MiniScrub as a tool for preprocessing Nanopore reads for downstream analyses. MiniScrub is open-source software and is available at https://bitbucket.org/berkeleylab/jgi-miniscrub

scholarly journals NGSpeciesID: DNA barcode and amplicon consensus generation from long-read sequencing data

2020 ◽  
Author(s):  
Kristoffer Sahlin ◽  
Marisa Lim ◽  
Stefan Prost
Keyword(s):  
High Throughput Sequencing ◽  
Dna Barcode ◽  
Amplicon Sequencing ◽  
Error Rates ◽  
Sequencing Data ◽  
Sequencing Platform ◽  
Consensus Sequences ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read

Third generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have gained popularity over the last years. These platforms can generate millions of long read sequences. This is not only advantageous for genome sequencing projects, but also for amplicon-based high-throughput sequencing experiments, such as DNA barcoding. However, the relatively high error rates associated with these technologies still pose challenges for generating high quality consensus sequences. Here we present NGSpeciesID, a program which can generate highly accurate consensus sequences from long-read amplicon sequencing technologies, including ONT and PacBio. The tool includes clustering of the reads to help filter out contaminants or reads with high error rates and employs polishing strategies specific to the appropriate sequencing platform. We show that NGSpeciesID produces consensus sequences with improved usability by minimizing preprocessing and software installation and scalability by enabling rapid processing of hundreds to thousands of samples, while maintaining similar consensus accuracy as current pipelines

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