scholarly journals Time course profiling of host cell response to herpesvirus infection using nanopore and synthetic long-read transcriptome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zoltán Maróti ◽  
Dóra Tombácz ◽  
Norbert Moldován ◽  
Gábor Torma ◽  
Victoria A. Jefferson ◽  
...  
Keyword(s):  
Gene Networks ◽  
Time Course ◽  
Host Cells ◽  
Nanopore Sequencing ◽  
Splice Isoforms ◽  
Transcript Isoforms ◽  
First Report ◽  
Host Cell Response ◽  
Long Read

AbstractThird-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcript isoforms identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection causes differential expression of host transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic transcriptomes in any organisms.

scholarly journals Long-read Time-course Profiling of the Host Cell Response to Herpesvirus Infection Using Nanopore and Synthetic Long-Read Transcriptome Sequencing 

2021 ◽  
Author(s):  
Zoltán Maróti ◽  
Norbert Moldován ◽  
Gábor Torma ◽  
Victoria A. Jefferson ◽  
Zsolt Csabai ◽  
...  
Keyword(s):  
Gene Networks ◽  
Time Course ◽  
Average Distance ◽  
Host Cells ◽  
Nanopore Sequencing ◽  
Splice Isoforms ◽  
Transcript Isoforms ◽  
First Report ◽  
Long Read

Abstract Third-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcripts identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection does not lead to a change in the average distance between promoters and transcription start sites, and between polyadenylation signals and transcription end sites. However, it causes differential expression of transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic changes of transcriptomes in any organisms.

scholarly journals Time-course transcriptome analysis of host cell response to poxvirus infection using a dual long-read sequencing approach

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Zoltán Maróti ◽  
Dóra Tombácz ◽  
István Prazsák ◽  
Norbert Moldován ◽  
Zsolt Csabai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Time Course ◽  
Time Lapse ◽  
Host Gene Expression ◽  
Transcript Isoforms ◽  
Short Read Sequencing ◽  
Host Cell Response ◽  
Long Read ◽  
Novel Transcripts ◽  
Transcriptional Start Sites

Abstract Objective In this study, we applied two long-read sequencing (LRS) approaches, including single-molecule real-time and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of host gene expression as a response to Vaccinia virus infection. Transcriptomes determined using short-read sequencing approaches are incomplete because these platforms are inefficient or fail to distinguish between polycistronic RNAs, transcript isoforms, transcriptional start sites, as well as transcriptional readthroughs and overlaps. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Results In this work, we identified a number of novel transcripts and transcript isoforms of Chlorocebus sabaeus. Additionally, analysis of the most abundant 768 host transcripts revealed a significant overrepresentation of the class of genes in the “regulation of signaling receptor activity” Gene Ontology annotation as a result of viral infection.

scholarly journals Metagenomic characterization of a harmful algal bloom using nanopore sequencing

2020 ◽  
Author(s):  
Peter W. Schafran ◽  
Victor Cai ◽  
Hsiao-Pei Yang ◽  
Fay-Wei Li
Keyword(s):  
Harmful Algal Blooms ◽  
Algal Blooms ◽  
Harmful Algal Bloom ◽  
Environmental Data ◽  
Nanopore Sequencing ◽  
Current Trends ◽  
Oxford Nanopore ◽  
Single Genome ◽  
Long Read

ABSTRACTWater bodies around the world are increasingly threatened by harmful algal blooms (HABs) under current trends of rising water temperature and nutrient load. Metagenomic characterization of HABs can be combined with water quality and environmental data to better understand and predict the occurrence of toxic events. However, standard short-read sequencing typically yields highly fragmented metagenomes, preventing direct connection of genes to a single genome. Using Oxford Nanopore long-read sequencing, we were able to obtain high quality metagenome-assembled genomes, and show that dominant organisms in a HAB are readily identified, though different analyses disagreed on the identity of rare taxa. Genes from diverse functional categories were found not only in the most dominant genera, but also in several less common ones. Using simulated datasets, we show that the Flongle flowcell may provide an option for HAB monitoring with less data, at the expense of failing to detect rarer organisms and increasing fragmentation of the metagenome. Based on these results, we believe that Nanopore sequencing provides a fast, portable, and affordable method for studying HABs.

Assessing the utility of long-read nanopore sequencing for rapid and efficient characterization of mobile element insertions

2020 ◽  
Author(s):  
Christopher M. Watson ◽  
Laura A. Crinnion ◽  
Helen Lindsay ◽  
Rowena Mitchell ◽  
Nick Camm ◽  
...  
Keyword(s):  
Mobile Element ◽  
Nanopore Sequencing ◽  
Long Read

scholarly journals Time-course profiling of bovine alphaherpesvirus 1.1 transcriptome using multiplatform sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Norbert Moldován ◽  
Gábor Torma ◽  
Gábor Gulyás ◽  
Ákos Hornyák ◽  
Zoltán Zádori ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Time Course ◽  
Splice Variants ◽  
Library Preparation ◽  
Transcription Start ◽  
Viral Mrnas ◽  
Transcript Isoforms ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies

AbstractLong-read sequencing (LRS) has become a standard approach for transcriptome analysis in recent years. Bovine alphaherpesvirus 1 (BoHV-1) is an important pathogen of cattle worldwide. This study reports the profiling of the dynamic lytic transcriptome of BoHV-1 using two long-read sequencing (LRS) techniques, the Oxford Nanopore Technologies MinION, and the LoopSeq synthetic LRS methods, using multiple library preparation protocols. In this work, we annotated viral mRNAs and non-coding transcripts, and a large number of transcript isoforms, including transcription start and end sites, as well as splice variants of BoHV-1. Our analysis demonstrated an extremely complex pattern of transcriptional overlaps.

scholarly journals Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing

2021 ◽  
Author(s):  
Matthew J. Tarnowski ◽  
Thomas E. Gorochowski
Keyword(s):  
Rna Sequencing ◽  
Massively Parallel ◽  
Dna Assembly ◽  
Sequence Context ◽  
One Pot ◽  
Transcript Isoforms ◽  
Long Read ◽  
Nucleotide Resolution

AbstractTranscriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as ‘transcriptional valves’ which can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly we construct 1183 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to simultaneously characterize the entire pool at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate their function using nearby sequence context. This work provides new avenues for controlling transcription and demonstrates the value of long-read sequencing for exploring complex sequence-function landscapes.

scholarly journals Time-Course Transcriptome Profiling of a Poxvirus Using Long-Read Full-Length Assay

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 919
Author(s):  
Dóra Tombácz ◽  
István Prazsák ◽  
Gábor Torma ◽  
Zsolt Csabai ◽  
Zsolt Balázs ◽  
...  
Keyword(s):  
Single Molecule ◽  
Time Course ◽  
Transcriptome Profiling ◽  
Time Lapse ◽  
Full Length ◽  
Read Length ◽  
Transcript Isoforms ◽  
Long Read ◽  
Second Generation Sequencing ◽  
Transcriptional Start Sites

Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of VACV gene expression.

scholarly journals Identification and Characterization of Inorganic Pyrophosphatase and PAP Phosphatase from Thermococcus onnurineus NA1

2009 ◽  
Vol 191 (10) ◽  
pp. 3415-3419 ◽  
Author(s):  
Hyun Sook Lee ◽  
Yun Jae Kim ◽  
Jung-Hyun Lee ◽  
Sung Gyun Kang
Keyword(s):  
Gene Clusters ◽  
Inorganic Pyrophosphatase ◽  
Thermococcus Onnurineus Na1 ◽  
First Report ◽  
Activation System ◽  
Sulfate Activation ◽  
Pyrococcus Abyssi ◽  
Identification And Characterization ◽  
Hypothetical Genes

ABSTRACT Two hypothetical genes were functionally verified to be a pyrophosphatase and a PAP phosphatase in Thermococcus onnurineus NA1. This is the first report of the pyrophosphatases and the PAP phosphatases being organized in the gene clusters of the sulfate activation system only in T. onnurineus NA1 and “Pyrococcus abyssi.”

scholarly journals MBRS-47. RAPID MOLECULAR SUBGROUPING OF MEDULLOBLASTOMA BASED ON DNA METHYLATION BY NANOPORE SEQUENCING

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Julien Masliah-Planchon ◽  
Elodie Girard ◽  
Philipp Euskirchen ◽  
Christine Bourneix ◽  
Delphine Lequin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Molecule ◽  
Nanopore Sequencing ◽  
Molecular Subgroup ◽  
Group Assignment ◽  
Group 4 ◽  
Methylation Assay ◽  
Tumor Group ◽  
Long Read ◽  
Group 3

Abstract Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples. Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature; 2018) to generate a score that estimates a confidence with a tumor group assignment. Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay. In conclusion, we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

scholarly journals Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq

2021 ◽  
Author(s):  
Martin Philpott ◽  
Jonathan Watson ◽  
Anjan Thakurta ◽  
Tom Brown ◽  
Tom Brown ◽  
...  
Keyword(s):  
Single Cell ◽  
Error Detection ◽  
Single Cells ◽  
Fusion Transcript ◽  
Building Blocks ◽  
Myeloma Cell ◽  
Nanopore Sequencing ◽  
Long Read ◽  
Unique Molecular Identifier ◽  
Transcript Detection

AbstractHere we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

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