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

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.

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

10.1101/2021.12.17.457386 ◽

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.

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

10.21203/rs.3.rs-637036/v1 ◽

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.

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

10.21203/rs.3.rs-17068/v2 ◽

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.

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scFlow: A Scalable and Reproducible Analysis Pipeline for Single-Cell RNA Sequencing Data

10.1101/2021.08.16.456499 ◽

2021 ◽

Author(s):

Combiz Khozoie ◽

Nurun Fancy ◽

Mahdi Moradi Marjaneh ◽

Alan E. Murphy ◽

Paul M. Matthews ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Sparse Matrix ◽

Data Generation ◽

Sequencing Data ◽

Alternative Analysis ◽

Analysis Pipeline ◽

Matrix Quality ◽

Single Cell Rna Sequencing ◽

Public Datasets

Advances in single-cell RNA-sequencing technology over the last decade have enabled exponential increases in throughput: datasets with over a million cells are becoming commonplace. The burgeoning scale of data generation, combined with the proliferation of alternative analysis methods, led us to develop the scFlow toolkit and the nf-core/scflow pipeline for reproducible, efficient, and scalable analyses of single-cell and single-nuclei RNA-sequencing data. The scFlow toolkit provides a higher level of abstraction on top of popular single-cell packages within an R ecosystem, while the nf-core/scflow Nextflow pipeline is built within the nf-core framework to enable compute infrastructure-independent deployment across all institutions and research facilities. Here we present our flexible pipeline, which leverages the advantages of containerization and the potential of Cloud computing for easy orchestration and scaling of the analysis of large case/control datasets by even non-expert users. We demonstrate the functionality of the analysis pipeline from sparse-matrix quality control through to insight discovery with examples of analysis of four recently published public datasets and describe the extensibility of scFlow as a modular, open-source tool for single-cell and single nuclei bioinformatic analyses.

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

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.1035 ◽

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.

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Genome-Wide Identification and Characterization of Long Non-Coding RNAs in Peanut

Genes ◽

10.3390/genes10070536 ◽

2019 ◽

Vol 10 (7) ◽

pp. 536 ◽

Cited By ~ 2

Author(s):

Xiaobo Zhao ◽

Liming Gan ◽

Caixia Yan ◽

Chunjuan Li ◽

Quanxi Sun ◽

...

Keyword(s):

Large Scale ◽

Target Genes ◽

Sequencing Data ◽

Regulatory Processes ◽

Genome Wide ◽

Non Coding Rnas ◽

Identification And Characterization ◽

Lower Expression ◽

Weighted Correlation

Long non-coding RNAs (lncRNAs) are involved in various regulatory processes although they do not encode protein. Presently, there is little information regarding the identification of lncRNAs in peanut (Arachis hypogaea Linn.). In this study, 50,873 lncRNAs of peanut were identified from large-scale published RNA sequencing data that belonged to 124 samples involving 15 different tissues. The average lengths of lncRNA and mRNA were 4335 bp and 954 bp, respectively. Compared to the mRNAs, the lncRNAs were shorter, with fewer exons and lower expression levels. The 4713 co-expression lncRNAs (expressed in all samples) were used to construct co-expression networks by using the weighted correlation network analysis (WGCNA). LncRNAs correlating with the growth and development of different peanut tissues were obtained, and target genes for 386 hub lncRNAs of all lncRNAs co-expressions were predicted. Taken together, these findings can provide a comprehensive identification of lncRNAs in peanut.

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Swan: a library for the analysis and visualization of long-read transcriptomes

Bioinformatics ◽

10.1093/bioinformatics/btaa836 ◽

2020 ◽

Author(s):

Fairlie Reese ◽

Ali Mortazavi

Keyword(s):

Rna Sequencing ◽

Cell Lines ◽

Intron Retention ◽

Exon Skipping ◽

Differentially Expressed ◽

Transcript Isoforms ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read

Abstract Motivation Long-read RNA-sequencing technologies such as PacBio and Oxford Nanopore have discovered an explosion of new transcript isoforms that are difficult to visually analyze using currently available tools. We introduce the Swan Python library, which is designed to analyze and visualize transcript models. Results Swan finds 4909 differentially expressed transcripts between cell lines HepG2 and HFFc6, including 279 that are differentially expressed even though the parent gene is not. Additionally, Swan discovers 285 reproducible exon skipping and 47 intron retention events not recorded in the GENCODE v29 annotation. Availability and implementation The Swan library for Python 3 is available on PyPi at https://pypi.org/project/swan-vis/ and on GitHub at https://github.com/mortazavilab/swan_vis.

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

Microbiology Resource Announcements ◽

10.1128/mra.00818-19 ◽

2019 ◽

Vol 8 (34) ◽

Cited By ~ 1

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.

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