scholarly journals isoformant: A visual toolkit for reference-free long-read isoform analysis at single-read resolution

2021 ◽  
Author(s):  
Daniel D Le ◽  
Faye T Orcales ◽  
William Stephenson
Keyword(s):  
Region Of Interest ◽  
Sequencing Data ◽  
Consensus Sequences ◽  
Interactive Analysis ◽  
Isoform Diversity ◽  
Oxford Nanopore ◽  
Long Read ◽  
Multiple Samples ◽  
Read Distribution

isoformant is an analytical toolkit for isoform characterization of Oxford Nanopore Technologies (ONT) long-transcript sequencing data (i.e. direct RNA and cDNA). Deployment of these tools using Jupyter Notebook enables interactive analysis of user- defined region-of-interest (ROI), typically a gene. The core module of isoformant clus- ters sequencing reads by k-mer density to generate isoform consensus sequences without the requirement for a reference genome or prior annotations. The inclusion of differential isoform usage hypothesis testing based on read distribution among clusters enables com- parison across multiple samples. Here, as proof-of-principle, we demonstrate the utility of isoformant for analyzing isoform diversity of commercially-available isoform standard mixtures. isoformant is available here: https://github.com/danledinh/isoformant.

scholarly journals TagSeqTools: a flexible and comprehensive analysis pipeline for NAD tagSeq data

2020 ◽  
Author(s):  
Huan Zhong ◽  
Zongwei Cai ◽  
Zhu Yang ◽  
Yiji Xia
Keyword(s):  
Rna Sequencing ◽  
Comprehensive Analysis ◽  
Enzymatic Reactions ◽  
Computational Tool ◽  
Sequencing Data ◽  
Analysis Pipeline ◽  
Oxford Nanopore ◽  
Long Read ◽  
Identification And Characterization

AbstractNAD tagSeq has recently been developed for the identification and characterization of NAD+-capped RNAs (NAD-RNAs). This method adopts a strategy of chemo-enzymatic reactions to label the NAD-RNAs with a synthetic RNA tag before subjecting to the Oxford Nanopore direct RNA sequencing. A computational tool designed for analyzing the sequencing data of tagged RNA will facilitate the broader application of this method. Hence, we introduce TagSeqTools as a flexible, general pipeline for the identification and quantification of tagged RNAs (i.e., NAD+-capped RNAs) using long-read transcriptome sequencing data generated by NAD tagSeq method. TagSeqTools comprises two major modules, TagSeek for differentiating tagged and untagged reads, and TagSeqQuant for the quantitative and further characterization analysis of genes and isoforms. Besides, the pipeline also integrates some advanced functions to identify antisense or splicing, and supports the data reformation for visualization. Therefore, TagSeqTools provides a convenient and comprehensive workflow for researchers to analyze the data produced by the NAD tagSeq method or other tagging-based experiments using Oxford nanopore direct RNA sequencing. The pipeline is available at https://github.com/dorothyzh/TagSeqTools, under Apache License 2.0.

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

Dual Isoform Sequencing Reveals a Multifaceted Transcriptional Architecture of a Prototype Baculovirus

2021 ◽  
Author(s):  
Gábor Torma ◽  
Dóra Tombácz ◽  
Norbert Moldován ◽  
Ádám Fülöp ◽  
István Prazsák ◽  
...  
Keyword(s):  
Protein Coding ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Oxford Nanopore ◽  
The Pacific ◽  
Viral Genes ◽  
Long Read ◽  
Oxford Nanopore Technologies ◽  
Overlapping Transcripts

Abstract In this study, we used two long-read sequencing (LRS) techniques, Sequel from the Pacific Biosciences and MinION from Oxford Nanopore Technologies, for the transcriptional characterization of a prototype baculovirus, Autographacalifornica multiple nucleopolyhedrovirus. LRS is able to read full-length RNA molecules, and thereby to distinguish between transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts. Altogether, we detected 875 transcripts, of which 759 are novel and 116 have been annotated previously. These RNA molecules include 41 novel putative protein coding transcript (each containing 5’-truncated in-frame ORFs), 14 monocistronic transcripts, 99 multicistronic RNAs, 101 non-coding RNA, and 504 length isoforms. We also detected RNA methylation in 12 viral genes and RNA hyper-editing in the longer 5’-UTR transcript isoform of ORF 19 gene.

scholarly journals Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

2020 ◽  
Author(s):  
Michael Liem ◽  
Tonny Regensburg-Tuïnk ◽  
Christiaan Henkel ◽  
Hans Jansen ◽  
Herman Spaink
Keyword(s):  
Microbial Diversity ◽  
Environmental Samples ◽  
Sea Water ◽  
Flow Cells ◽  
Oxford Nanopore ◽  
Challenging Tasks ◽  
Long Read ◽  
Close Relatives ◽  
Oxford Nanopore Technologies

Abstract Objective: Currently the majority of non-culturable microbes in sea water are yet to be discovered, Nanopore offers a solution to overcome the challenging tasks to identify the genomes and complex composition of oceanic microbiomes. In this study we evaluate the utility of Oxford Nanopore Technologies (ONT) sequencing to characterize microbial diversity in seawater from multiple locations. We compared the microbial species diversity of retrieved environmental samples from two different locations and time points.Results: With only three ONT flow cells we were able to identify thousands of organisms, including bacteriophages, from which a large part at species level. It was possible to assemble genomes from environmental samples with Flye. In several cases this resulted in >1 Mbp contigs and in the particular case of a Thioglobus singularis species it even produced a near complete genome. k-mer analysis reveals that a large part of the data represents species of which close relatives have not yet been deposited to the database. These results show that our approach is suitable for scalable genomic investigations such as monitoring oceanic biodiversity and provides a new platform for education in biodiversity.

scholarly journals 1202. Multimodal Sequencing of a Clonal Case Cluster of Carbapenem-Resistant Citrobacter Reveals Unexpectedly Rapid Dynamics of KPC3-Containing Plasmids

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S364-S364
Author(s):  
Roby Bhattacharyya ◽  
Alejandro Pironti ◽  
Bruce J Walker ◽  
Abigail Manson ◽  
Virginia Pierce ◽  
...  
Keyword(s):  
Point Mutations ◽  
Illumina Miseq ◽  
Nucleotide Polymorphisms ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Carbapenem Resistant ◽  
Oxford Nanopore ◽  
Close Relationship ◽  
Long Read ◽  
Carbapenem Resistant Enterobacteriaceae

Abstract Background Carbapenem-resistant Enterobacteriaceae (CRE) are a major public health threat. We report four clonally related Citrobacter freundii isolates harboring the blaKPC-3 carbapenemase in April–May 2017 that are nearly identical to a strain from 2014 at the same institution. Despite differing by ≤5 single nucleotide polymorphisms (SNPs), these isolates exhibited dramatic differences in carbapenemase plasmid architecture. Methods We sequenced four carbapenem-resistant C. freundii isolates from 2017 and compared them with an ongoing CRE surveillance project at our institution. SNPs were identified from Illumina MiSeq data aligned to a reference genome using the variant caller Pilon. Plasmids were assembled from Illumina and Oxford Nanopore sequencing data using Unicycler. Results The four 2017 isolates differed from one another by 0–5 chromosomal SNPs; two were identical. With one exception, these isolates differed by >38,000 SNPs from 25 C. freundii isolates sequenced from 2013 to 2017 at the same institution for CRE surveillance. The exception was a 2014 isolate that differed by 13–16 SNPs from each 2017 isolate, with 13 SNPs common to all four. Each C. freundii isolate harbored wild-type blaKPC-3. Despite the close relationship among the 2017 cluster, the plasmids harboring the blaKPC-3 genes differed dramatically: the carbapenemase occurred in one of the two different plasmids, with rearrangements between these plasmids across isolates. The related 2014 isolate harbored both plasmids, each with a separate copy of blaKPC-3. No transmission chains were found between any of the affected patients. Conclusion WGS confirmed clonality among four contemporaneous blaKPC-3-containing C. freundii isolates, and marked similarity with a 2014 isolate, within an institution. That only 13–16 SNPs varied between the 2014 and 2017 isolates suggests durable persistence of the blaKPC-3 gene within this lineage in a hospital ecosystem. The plasmids harboring these carbapenemase genes proved remarkably plastic, with plasmid loss and rearrangements occurring on the same time scale as two to three chromosomal point mutations. Combining short and long-read sequencing in a case cluster uniquely revealed unexpectedly rapid dynamics of carbapenemase plasmids, providing critical insight into their manner of spread. Disclosures M. J. Ferraro, SeLux Diagnostics: Scientific Advisor and Shareholder, Consulting fee. D. C. Hooper, SeLux Diagnostics: Scientific Advisor, Consulting fee.

scholarly journals Complete Genome Sequence of Rubrobacter xylanophilus Strain AA3-22, Isolated from Arima Onsen in Japan

2019 ◽  
Vol 8 (34) ◽  
Author(s):  
Natsuki Tomariguchi ◽  
Kentaro Miyazaki
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Hot Spring ◽  
Sequencing Data ◽  
Short Read ◽  
Content Type ◽  
Short Read Sequencing ◽  
Oxford Nanopore ◽  
Long Read

Rubrobacter xylanophilus strain AA3-22, belonging to the phylum Actinobacteria, was isolated from nonvolcanic Arima Onsen (hot spring) in Japan. Here, we report the complete genome sequence of this organism, which was obtained by combining Oxford Nanopore long-read and Illumina short-read sequencing data.

scholarly journals Characterization of mRNA isoform diversity in a transgenic model of tau pathology using targeted long‐read sequencing

2020 ◽  
Vol 16 (S3) ◽  
Author(s):  
Szi Kay Leung ◽  
Aaron Jeffries ◽  
Eilis Hannon ◽  
Isabel Castanho ◽  
Karen Moore ◽  
...  
Keyword(s):  
Tau Pathology ◽  
Transgenic Model ◽  
Isoform Diversity ◽  
Long Read ◽  
Mrna Isoform

scholarly journals Plasmidome analysis of carbapenem-resistant Enterobacteriaceae isolated in Vietnam

2020 ◽  
Author(s):  
Aki Hirabayashi ◽  
Koji Yahara ◽  
Satomi Mitsuhashi ◽  
So Nakagawa ◽  
Tadashi Imanishi ◽  
...  
Keyword(s):  
Carbapenem Resistance ◽  
Genomic Epidemiology ◽  
Carbapenem Resistant ◽  
Oxford Nanopore ◽  
Carbapenemase Gene ◽  
Long Read ◽  
Severe Infections ◽  
Oxford Nanopore Technologies ◽  
Carbapenem Resistant Enterobacteriaceae

Carbapenem-resistant Enterobacteriaceae (CRE) represent a serious threat to public health due to limited management of severe infections and high mortality. The rate of resistance of Enterobacteriaceae isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene bla NDM-1 , spread via plasmids among Enterobacteriaceae in Vietnam. In this study, we performed detection and molecular characterization of bla NDM-1 -carrying plasmids in CRE isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-resistant isolates from Enterobacteriaceae clinically isolated in a reference medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 12 isolates harboring bla NDM-1 were sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to bla NDM-1 , leading to multidrug resistance of their bacterial hosts. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.

scholarly journals Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

2021 ◽  
Vol 12 ◽  
Author(s):  
Davide Bolognini ◽  
Alberto Magi
Keyword(s):  
Variant Calling ◽  
Research Report ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Factors Affecting ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Sequencing Studies ◽  
Long Read

Structural variants (SVs) are genomic rearrangements that involve at least 50 nucleotides and are known to have a serious impact on human health. While prior short-read sequencing technologies have often proved inadequate for a comprehensive assessment of structural variation, more recent long reads from Oxford Nanopore Technologies have already been proven invaluable for the discovery of large SVs and hold the potential to facilitate the resolution of the full SV spectrum. With many long-read sequencing studies to follow, it is crucial to assess factors affecting current SV calling pipelines for nanopore sequencing data. In this brief research report, we evaluate and compare the performances of five long-read SV callers across four long-read aligners using both real and synthetic nanopore datasets. In particular, we focus on the effects of read alignment, sequencing coverage, and variant allele depth on the detection and genotyping of SVs of different types and size ranges and provide insights into precision and recall of SV callsets generated by integrating the various long-read aligners and SV callers. The computational pipeline we propose is publicly available at https://github.com/davidebolo1993/EViNCe and can be adjusted to further evaluate future nanopore sequencing datasets.

scholarly journals Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

2020 ◽  
Author(s):  
Michael Liem ◽  
A.J.G. Regensburg-Tuïnk ◽  
C.V. Henkel ◽  
H.P. Spaink
Keyword(s):  
Microbial Diversity ◽  
Environmental Samples ◽  
Sea Water ◽  
Flow Cells ◽  
Oxford Nanopore ◽  
Challenging Tasks ◽  
Long Read ◽  
Close Relatives ◽  
Oxford Nanopore Technologies

Abstract Objective Currently the majority of non-culturable microbes in sea water are yet to be discovered, Nanopore offers a solution to overcome the challenging tasks to identify the genomes and complex composition of oceanic microbiomes. In this study we evaluate the utility of Oxford Nanopore Technologies (ONT) sequencing to characterize microbial diversity in seawater from multiple locations. We compared the microbial species diversity of retrieved environmental samples from two different locations and time points. Results With only three ONT flow cells we were able to identify thousands of organisms, including bacteriophages, from which a large part at species level. It was possible to assemble genomes from environmental samples with Flye. In several cases this resulted in >1 Mbp contigs and in the particular case of a Thioglobus singularis species it even produced a near complete genome. k-mer analysis reveals that a large part of the data represents species of which close relatives have not yet been deposited to the database. These results show that our approach is suitable for scalable genomic investigations such as monitoring oceanic biodiversity and provides a new platform for education in biodiversity.

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