scholarly journals Long-read Sequencing Uncovers a Complex Transcriptome Topology in Varicella Zoster Virus

2018 ◽  
Author(s):  
István Prazsák ◽  
Norbert Moldován ◽  
Dóra Tombácz ◽  
Klára Megyeri ◽  
Attila Szűcs ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Genomic Region ◽  
Transcript Isoforms ◽  
Sequencing Platform ◽  
Varicella Zoster ◽  
Rna Molecules ◽  
Oxford Nanopore ◽  
Complex Structural Variation ◽  
Long Read ◽  
Complex Structural

AbstractBackgroundVaricella zoster virus (VZV) is a human pathogenic alphaherpesvirus harboring a relatively large DNA molecule. The VZV transcriptome has already been analyzed by microarray and short-read sequencing analyses. However, both approaches have substantial limitations when used for structural characterization of transcript isoforms, even if supplemented with primer extension or other techniques. Among others, they are inefficient in distinguishing between embedded RNA molecules, transcript isoforms, including splice and length variants, as well as between alternative polycistronic transcripts. It has been demonstrated in several studies that long-read sequencing is able to circumvent these problems.ResultsIn this work, we report the analysis of VZV lytic transcriptome using the Oxford Nanopore Technologies sequencing platform. These investigations have led to the identification of 114 novel transcripts, including mRNAs, non-coding RNAs, polycistronic RNAs and complex transcripts, as well as 10 novel spliced transcripts and 27 novel transcription start site isoforms and transcription end site isoforms. A novel class of transcripts, the nroRNAs are described in this study. These transcripts are encoded by the genomic region located in close vicinity to the viral replication origin. We also show that the VZV latency transcript (VLT) exhibits a more complex structural variation than formerly believed. Additionally, we have detected RNA editing in a novel non-coding RNA molecule.ConclusionsOur investigations disclosed a composite transcriptomic architecture of VZV, including the discovery of novel RNA molecules and transcript isoforms, as well as a complex meshwork of transcriptional read-throughs and overlaps. The results represent a substantial advance in the annotation VZV transcriptome and in understanding the molecular biology of the herpesviruses in general.

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 Lytic Transcriptome Dataset of Varicella Zoster Virus Generated by Long-Read Sequencing

2018 ◽  
Vol 9 ◽  
Author(s):  
Dóra Tombácz ◽  
István Prazsák ◽  
Norbert Moldován ◽  
Attila Szűcs ◽  
Zsolt Boldogkői
Keyword(s):  
Varicella Zoster Virus ◽  
Varicella Zoster ◽  
Long Read

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

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.

scholarly journals Multiple Long-read Sequencing Survey of Herpes Simplex Virus Lytic Transcriptome

2019 ◽  
Author(s):  
Dóra Tombácz ◽  
Zsolt Balázs ◽  
Gábor Gulyás ◽  
Zsolt Csabai ◽  
Miklós Boldogkoi ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Single Molecule ◽  
Rna Molecules ◽  
Oxford Nanopore ◽  
Dna Strands ◽  
Long Read ◽  
Simplex Virus ◽  
Transcriptional Start Sites ◽  
Hsv 1

ABSTRACTLong-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying transcript isoforms including polycistronic and multi-spliced RNA molecules, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 160 previously undetected transcripts, including coding and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we determined previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5’ regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.

scholarly journals Parallel and scalable workflow for the analysis of Oxford Nanopore direct RNA sequencing datasets

2019 ◽  
Author(s):  
Luca Cozzuto ◽  
Huanle Liu ◽  
Leszek P. Pryszcz ◽  
Toni Hermoso Pulido ◽  
Julia Ponomarenko ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Single Molecule ◽  
Tail Length ◽  
Rna Modification ◽  
Sequencing Data ◽  
Polya Tail ◽  
Sequencing Platform ◽  
Rna Molecules ◽  
Oxford Nanopore ◽  
Quality Filtering

ABSTRACTThe direct RNA sequencing platform offered by Oxford Nanopore Technologies allows for direct measurement of RNA molecules without the need of conversion to complementary DNA, fragmentation or amplification. As such, it is virtually capable of detecting any given RNA modification present in the molecule that is being sequenced, as well as provide polyA tail length estimations at the level of individual RNA molecules. Although this technology has been publicly available since 2017, the complexity of the raw Nanopore data, together with the lack of systematic and reproducible pipelines, have greatly hindered the access of this technology to the general user. Here we address this problem by providing a fully benchmarked workflow for the analysis of direct RNA sequencing reads, termed MasterOfPores. The pipeline converts raw current intensities into multiple types of processed data, providing metrics of the quality of the run, quality-filtering, base-calling and mapping. The output of the pipeline can in turn be used to compute per-gene counts, RNA modifications, and prediction of polyA tail length and RNA isoforms. The software is written using the NextFlow framework for parallelization and portability, and relies on Linux containers such as Docker and Singularity for achieving better reproducibility. The MasterOfPores workflow can be executed on any Unix-compatible OS on a computer, cluster or cloud without the need of installing any additional software or dependencies, and is freely available in Github (https://github.com/biocorecrg/master_of_pores). This workflow will significantly simplify the analysis of nanopore direct RNA sequencing data by non-bioinformatics experts, thus boosting the understanding of the (epi)transcriptome with single molecule resolution.

scholarly journals PacBio library preparation using blunt-end adapter ligation produces significant artefactual fusion DNA sequences

2018 ◽  
Author(s):  
Paul Griffith ◽  
Castle Raley ◽  
David Sun ◽  
Yongmei Zhao ◽  
Zhonghe Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Turnaround Time ◽  
Library Preparation ◽  
Adapter Ligation ◽  
Sequence Production ◽  
Long Reads ◽  
Complex Structural Variation ◽  
Long Read ◽  
Complex Structural

AbstractPacific Biosciences’ (PacBio) RS II sequencer, utilizing Single-Molecule, Real-Time (SMRT) technology, has revolutionized next-generation sequencing by providing an accurate long-read platform. PacBio single-molecule long reads have been used to delineate complex spliceoforms, detect mutations in highly homologous sequences, identify mRNA chimeras and chromosomal translocations, accurately haplotype phasing over multiple kilobase distances and aid in assembly of genomes with complex structural variation. The PacBio protocol for preparation of sequencing templates employs blunt-end hairpin adapter ligation, which enables a short turnaround time for sequence production. However, we have found a significant portion of sequencing yield contains chimeric reads resulting from blunt-end ligation of multiple template molecules to each other prior to adapter ligation. These artefactual fusion DNA sequences pose a major challenge to analysis and can lead to false-positive detection of fusion events. We assessed the frequency of artefactual fusion when using blunt-end adapter ligation and compared it to an alternative method using A/T overhang adapter ligation. The A/T overhang adapter ligation method showed a vast improvement in limiting artefactual fusion events and is now our recommended procedure for adapter ligation during PacBio library preparation.

scholarly journals Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus

BMC Genomics ◽  
2018 ◽  
Vol 19 (1) ◽  
Author(s):  
István Prazsák ◽  
Norbert Moldován ◽  
Zsolt Balázs ◽  
Dóra Tombácz ◽  
Klára Megyeri ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Varicella Zoster ◽  
Long Read

scholarly journals Transcriptome Profiling of Epstein-Barr Virus Using Nanopore Sequencing

2020 ◽  
Author(s):  
Norbert Moldován ◽  
Kálmán Szenthe ◽  
Ferenc Bánáti ◽  
Ádám Fülöp ◽  
Zsolt Csabai ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Transcriptome Profiling ◽  
Open Reading Frames ◽  
Splice Isoforms ◽  
Rna Molecules ◽  
Barr Virus ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Epstein Barr

Abstract 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- and Pacific Biosciences RS II-based long-read sequencing technologies. In this work, we use the Oxford Nanopore Technologies MinION platform for the characterization of the EBV transcriptomic architecture. Both amplified and non-amplified cDNA sequencings were applied for the generation of transcription 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. This study detected novel short genes (embedded into longer host genes) containing 5’-truncated in-frame open reading frames (ORFs), which might 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 Ori-associated RNA molecules. Additionally, novel mono- and polycistronic, as well as complex transcripts have been uncovered. An intricate meshwork of transcriptional overlaps has also been revealed.

scholarly journals Combined short and long-read sequencing reveals a complex transcriptomic architecture of African swine fever virus

2020 ◽  
Author(s):  
Gábor Torma ◽  
Dóra Tombácz ◽  
Zsolt Csabai ◽  
Norbert Moldován ◽  
István Mészáros ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
African Swine Fever Virus ◽  
Genetic Regulation ◽  
African Swine Fever ◽  
Fever Virus ◽  
Current View ◽  
Preparation Methods ◽  
Rna Molecules ◽  
Oxford Nanopore ◽  
Long Read

ABSTRACTAfrican swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Understanding of genetic regulation provides new insights into the virus pathogenicity, which can help prevent epidemics. Short-read sequencing (SRS) is able to produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for the comprehensive annotation of transcriptomes. Long-read sequencing (LRS) is able to overcome some of the limitations of SRS, but they also have drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the transcriptomic atlas of ASFV. This work identified a large number of novel genes, transcripts and RNA isoforms, and annotated the precise termini of previously described RNA molecules. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism. A multifaceted meshwork of transcriptional overlaps is also discovered.

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