scholarly journals Single-Molecule Real-Time Sequencing of the Madhuca pasquieri (Dubard) Lam. Transcriptome Reveals the Diversity of Full-Length Transcripts

Forests ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 866
Author(s):  
Lei Kan ◽  
Qicong Liao ◽  
Zhiyao Su ◽  
Yushan Tan ◽  
Shuyu Wang ◽  
...  
Keyword(s):  
Seed Germination ◽  
Single Molecule ◽  
Developmental Stages ◽  
De Novo ◽  
Full Length ◽  
Wild Plant ◽  
Transcript Isoforms ◽  
Long Read ◽  
Full Length Transcript ◽  
Generation Sequencing

Madhuca pasquieri (Dubard) Lam. is a tree on the International Union for Conservation of Nature Red List and a national key protected wild plant (II) of China, known for its seed oil and timber. However, lacking of genomic and transcriptome data for this species hampers study of its reproduction, utilization, and conservation. Here, single-molecule long-read sequencing (PacBio) and next-generation sequencing (Illumina) were combined to obtain the transcriptome from five developmental stages of M. pasquieri. Overall, 25,339 transcript isoforms were detected by PacBio, including 24,492 coding sequences (CDSs), 9440 simple sequence repeats (SSRs), 149 long non-coding RNAs (lncRNAs), and 182 alternative splicing (AS) events, a majority was retained intron (RI). A further 1058 transcripts were identified as transcriptional factors (TFs) from 51 TF families. PacBio recovered more full-length transcript isoforms with a longer length, and a higher expression level, whereas larger number of transcripts (124,405) was captured in de novo from Illumina. Using Nr, Swissprot, KOG, and KEGG databases, 24,405 transcripts (96.31%) were annotated by PacBio. Functional annotation revealed a role for the auxin, abscisic acid, gibberellin, and cytokinine metabolic pathways in seed germination and post-germination. These findings support further studies on seed germination mechanism and genome of M. pasquieri, and better protection of this endangered species.

scholarly journals ISOdb: A Comprehensive Database of Full-Length Isoforms Generated by Iso-Seq

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Shang-Qian Xie ◽  
Yue Han ◽  
Xiao-Zhou Chen ◽  
Tai-Yu Cao ◽  
Kai-Kai Ji ◽  
...  
Keyword(s):  
Single Molecule ◽  
Full Length ◽  
Public Access ◽  
Transcript Isoforms ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Depth Analysis ◽  
Gene Level ◽  
Long Read ◽  
Full Length Transcript

The accurate landscape of transcript isoforms plays an important role in the understanding of gene function and gene regulation. However, building complete transcripts is very challenging for short reads generated using next-generation sequencing. Fortunately, isoform sequencing (Iso-Seq) using single-molecule sequencing technologies, such as PacBio SMRT, provides long reads spanning entire transcript isoforms which do not require assembly. Therefore, we have developed ISOdb, a comprehensive resource database for hosting and carrying out an in-depth analysis of Iso-Seq datasets and visualising the full-length transcript isoforms. The current version of ISOdb has collected 93 publicly available Iso-Seq samples from eight species and presents the samples in two levels: (1) sample level, including metainformation, long read distribution, isoform numbers, and alternative splicing (AS) events of each sample; (2) gene level, including the total isoforms, novel isoform number, novel AS number, and isoform visualisation of each gene. In addition, ISOdb provides a user interface in the website for uploading sample information to facilitate the collection and analysis of researchers’ datasets. Currently, ISOdb is the first repository that offers comprehensive resources and convenient public access for hosting, analysing, and visualising Iso-Seq data, which is freely available.

scholarly journals Single-Molecule Long-Read Sequencing Reveals the Diversity of Full-Length Transcripts in Leaves of Gnetum (Gnetales)

2019 ◽  
Vol 20 (24) ◽  
pp. 6350 ◽  
Author(s):  
Nan Deng ◽  
Chen Hou ◽  
Fengfeng Ma ◽  
Caixia Liu ◽  
Yuxin Tian
Keyword(s):  
Single Molecule ◽  
Developmental Stages ◽  
Alternative Polyadenylation ◽  
Full Length ◽  
Stomatal Development ◽  
Rna Seq ◽  
Leaf Transcriptome ◽  
Long Read ◽  
Non Coding Rnas ◽  
A Site

The limitations of RNA sequencing make it difficult to accurately predict alternative splicing (AS) and alternative polyadenylation (APA) events and long non-coding RNAs (lncRNAs), all of which reveal transcriptomic diversity and the complexity of gene regulation. Gnetum, a genus with ambiguous phylogenetic placement in seed plants, has a distinct stomatal structure and photosynthetic characteristics. In this study, a full-length transcriptome of Gnetum luofuense leaves at different developmental stages was sequenced with the latest PacBio Sequel platform. After correction by short reads generated by Illumina RNA-Seq, 80,496 full-length transcripts were obtained, of which 5269 reads were identified as isoforms of novel genes. Additionally, 1660 lncRNAs and 12,998 AS events were detected. In total, 5647 genes in the G. luofuense leaves had APA featured by at least one poly(A) site. Moreover, 67 and 30 genes from the bHLH gene family, which play an important role in stomatal development and photosynthesis, were identified from the G. luofuense genome and leaf transcripts, respectively. This leaf transcriptome supplements the reference genome of G. luofuense, and the AS events and lncRNAs detected provide valuable resources for future studies of investigating low photosynthetic capacity of Gnetum.

scholarly journals High-throughput annotation of full-length long noncoding RNAs with Capture Long-Read Sequencing

2017 ◽  
Author(s):  
Julien Lagarde ◽  
Barbara Uszczynska-Ratajczak ◽  
Silvia Carbonell ◽  
SÍlvia Pérez-Lluch ◽  
Amaya Abad ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Noncoding Rnas ◽  
Splice Junction ◽  
Long Noncoding Rnas ◽  
Full Length ◽  
Novel Transcript ◽  
Mouse Tissues ◽  
Long Read ◽  
Full Length Transcript

AbstractAccurate annotations of genes and their transcripts is a foundation of genomics, but no annotation technique presently combines throughput and accuracy. As a result, reference gene collections remain incomplete: many gene models are fragmentary, while thousands more remain uncatalogued–particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), combining targeted RNA capture with third-generation long-read sequencing. We present an experimental re-annotation of the GENCODE intergenic lncRNA population in matched human and mouse tissues, resulting in novel transcript models for 3574 / 561 gene loci, respectively. CLS approximately doubles the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enable us to definitively characterize the genomic features of lncRNAs, including promoter- and gene-structure, and protein-coding potential. Thus CLS removes a longstanding bottleneck of transcriptome annotation, generating manual-quality full-length transcript models at high-throughput scales.Abbreviationsbpbase pairFLfull lengthntnucleotideROIread of insert, i.e. PacBio readSJsplice junctionSMRTsingle-molecule real-timeTMtranscript model

scholarly journals Long-read sequencing of Chrysanthemum morifolium cv. ‘Hangju’ transcriptome reveals flavonoid biosynthesis and regulation

2020 ◽  
Author(s):  
Tao Wang ◽  
Feng Yang ◽  
Qiaosheng Guo ◽  
Qingjun Zou ◽  
Wenyan Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Developmental Stages ◽  
Chrysanthemum Morifolium ◽  
Flavonoid Biosynthesis ◽  
Full Length ◽  
Bioactive Components ◽  
Smrt Sequencing ◽  
Major Genes ◽  
Long Read ◽  
Gene Expression Levels

Abstract Background: The inflorescence of Chrysanthemum morifolium cv. ‘Hangju’ has been widely used in China due to its antioxidant and anti-inflammatory properties. The biosynthesis and regulation of flavonoids, a group of bioactive components, in C. morifolium are poorly understood. Transcriptome sequencing is an effective method for obtaining transcript information. Therefore, single-molecule real-time (SMRT) sequencing was performed to obtain the full-length genes involved in flavonoid biosynthesis and regulation in C. morifolium.Results: High-quality RNA was extracted from the inflorescence of C. morifolium at different developmental stages and used to construct two libraries (0-5 kb and 4.5-10 kb) for sequencing. Finally, 125,532 non-redundant isoforms with a mean length of 2,009 bp were obtained. Of these, 2,083 transcripts were annotated to pathways related to flavonoid biosynthesis, and 56 isoforms were annotated as CHS, CHI, F3H, F3’H, FNS Ⅱ, FLS, DFR and ANS genes. Based on gene expression levels at different stages, we predicted the major genes involved in flavonoid biosynthesis. By phylogenetic analysis, we found two candidate MYB transcription factors (CmMYBF1 and CmMYBF2) activating flavonol biosynthesis.Conclusions: Based on the full-length transcriptomic data and further quantitative analysis, the major genes involved in flavonoid biosynthesis and regulation in C. morifolium were predicted in our study. The results provide a valuable theoretical basis for the introduction and cultivation of C. morifolium cv. ‘Hangju’.

scholarly journals Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chenchen Zhu ◽  
Jingyan Wu ◽  
Han Sun ◽  
Francesca Briganti ◽  
Benjamin Meder ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rna Binding ◽  
De Novo ◽  
Full Length ◽  
Binding Motif ◽  
Detection Accuracy ◽  
Transcript Isoforms ◽  
Cdna Sequences ◽  
Novel Transcript ◽  
Biological Interpretation

AbstractAlternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

scholarly journals Large-Scale Multiplexing Permits Full-Length Transcriptome Annotation of 32 Bovine Tissues From a Single Nanopore Flow Cell

2021 ◽  
Vol 12 ◽  
Author(s):  
Michelle M. Halstead ◽  
Alma Islas-Trejo ◽  
Daniel E. Goszczynski ◽  
Juan F. Medrano ◽  
Huaijun Zhou ◽  
...  
Keyword(s):  
Large Scale ◽  
Developmental Stages ◽  
Cell Types ◽  
Flow Cell ◽  
Full Length ◽  
Transcript Isoforms ◽  
Reference Transcript ◽  
Long Read ◽  
Additional Cell ◽  
Transcript Reconstruction

A comprehensive annotation of transcript isoforms in domesticated species is lacking. Especially considering that transcriptome complexity and splicing patterns are not well-conserved between species, this presents a substantial obstacle to genomic selection programs that seek to improve production, disease resistance, and reproduction. Recent advances in long-read sequencing technology have made it possible to directly extrapolate the structure of full-length transcripts without the need for transcript reconstruction. In this study, we demonstrate the power of long-read sequencing for transcriptome annotation by coupling Oxford Nanopore Technology (ONT) with large-scale multiplexing of 93 samples, comprising 32 tissues collected from adult male and female Hereford cattle. More than 30 million uniquely mapping full-length reads were obtained from a single ONT flow cell, and used to identify and characterize the expression dynamics of 99,044 transcript isoforms at 31,824 loci. Of these predicted transcripts, 21% exactly matched a reference transcript, and 61% were novel isoforms of reference genes, substantially increasing the ratio of transcript variants per gene, and suggesting that the complexity of the bovine transcriptome is comparable to that in humans. Over 7,000 transcript isoforms were extremely tissue-specific, and 61% of these were attributed to testis, which exhibited the most complex transcriptome of all interrogated tissues. Despite profiling over 30 tissues, transcription was only detected at about 60% of reference loci. Consequently, additional studies will be necessary to continue characterizing the bovine transcriptome in additional cell types, developmental stages, and physiological conditions. However, by here demonstrating the power of ONT sequencing coupled with large-scale multiplexing, the task of exhaustively annotating the bovine transcriptome – or any mammalian transcriptome – appears significantly more feasible.

scholarly journals Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5818 ◽  
Author(s):  
Jittima Piriyapongsa ◽  
Pavita Kaewprommal ◽  
Sirintra Vaiwsri ◽  
Songtham Anuntakarun ◽  
Warodom Wirojsirasak ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Energy Resource ◽  
Leaf Tissue ◽  
Full Length ◽  
Improvement Program ◽  
Sugarcane Cultivar ◽  
Reading Frame ◽  
Khon Kaen ◽  
Long Read

Background Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs. Methods We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors. Results A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants. Discussion The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

scholarly journals Single-molecule, full-length transcript isoform sequencing reveals disease mutation-associated RNA isoforms in cardiomyocytes

2021 ◽  
Author(s):  
Chenchen Zhu ◽  
Jingyan Wu ◽  
Han Sun ◽  
Francesca Briganti ◽  
Benjamin Meder ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rna Binding ◽  
De Novo ◽  
Full Length ◽  
Binding Motif ◽  
Detection Accuracy ◽  
Transcript Isoforms ◽  
Cdna Sequences ◽  
Novel Transcript ◽  
Biological Interpretation

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we established an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generated a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identified 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by novel IMMT isoforms. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms as the functional unit, instead of genes or exons, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

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 Long-read sequencing of Chrysanthemum morifolium cv. ‘Hangju’ transcriptome reveals flavonoid biosynthesis and regulation

2019 ◽  
Author(s):  
Tao Wang ◽  
Feng Yang ◽  
Qiaosheng Guo ◽  
Qingjun Zou ◽  
Wenyan Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Developmental Stages ◽  
Chrysanthemum Morifolium ◽  
Flavonoid Biosynthesis ◽  
Full Length ◽  
Bioactive Components ◽  
Smrt Sequencing ◽  
Major Genes ◽  
Long Read ◽  
Gene Expression Levels

Abstract Background: The inflorescence of Chrysanthemum morifolium cv. ‘Hangju’ has been widely used in China due to its antioxidant and anti-inflammatory properties. The biosynthesis and regulation of flavonoids, a group of bioactive components, in C. morifolium are poorly understood. Transcriptome sequencing is an effective method for obtaining transcript information. Therefore, single-molecule real-time (SMRT) sequencing was performed to obtain the full-length genes involved in flavonoid biosynthesis and regulation in C. morifolium. Results: High-quality RNA was extracted from the inflorescence of C. morifolium at different developmental stages and used to construct two libraries (0-5 kb and 4.5-10 kb) for sequencing. Finally, 125,532 non-redundant isoforms with a mean length of 2,009 bp were obtained. Of these, 2,083 transcripts were annotated to pathways related to flavonoid biosynthesis, and 56 isoforms were annotated as CHS, CHI, F3H, F3’H, FNS Ⅱ, FLS, DFR and ANS genes. Based on gene expression levels at different stages, we predicted the major genes involved in flavonoid biosynthesis. By phylogenetic analysis, we found two candidate MYB transcription factors (CmMYBF1 and CmMYBF2) activating flavonol biosynthesis. Conclusions: Based on the full-length transcriptomic data and further quantitative analysis, the major genes involved in flavonoid biosynthesis and regulation in C. morifolium were predicted in our study. The results provide a valuable theoretical basis for the introduction and cultivation of C. morifolium cv. ‘Hangju’.

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