Characterization of Full-Length Transcriptome Sequences and Splice Variants of Lateolabrax maculatus by Single-Molecule Long-Read Sequencing and Their Involvement in Salinity Regulation

AbstractState-of-the-art short-read transcriptome sequencing methods employ unique molecular identifier (UMI) to accurately classify and count mRNA transcripts. A fundamental limitation of UMI-based short-read transcriptome sequencing is that each read typically covers a small fraction of the transcript sequence. Efforts to accurately characterize splicing isoforms, arguably the largest source of variation in Human gene expression, using short read sequencing have therefore largely relied on computational predictions of transcript isoforms based on indirect observations. Here we describe a transcript counting, synthetic long read method for sequencing whole transcriptomes using short read sequencing platforms and no additional hardware. The method enables full-length mRNA sequence reconstruction at single-nucleotide resolutions with high-throughput, low error rates and UMI based transcript counting using any Illumina sequencer. We describe results from whole transcriptome sequencing from total RNA extracted from 3 human tissue samples: brain, liver, and blood. Reconstructed transcript sequences are characterized and annotated using SQANTI, an analysis pipeline for assessing the sequence quality of long-read transcriptomes. Our results demonstrate that LoopSeq synthetic long-read sequencing can reconstruct contigs up to 3,900nt full-length transcripts using tissue extracted RNA, as well as identify novel splice variants of known junction donors and acceptors.

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PacBio Single-Molecule Long-Read Sequencing Reveals Genes Tolerating Manganese Stress in Schima superba Saplings

Frontiers in Genetics ◽

10.3389/fgene.2021.635043 ◽

2021 ◽

Vol 12 ◽

Author(s):

Fiza Liaquat ◽

Muhammad Farooq Hussain Munis ◽

Samiah Arif ◽

Urooj Haroon ◽

Jianxin Shi ◽

...

Keyword(s):

Single Molecule ◽

Gene Annotation ◽

Treated Group ◽

Full Length ◽

Open Reading Frames ◽

Interacting Protein ◽

Schima Superba ◽

Long Read ◽

First Time ◽

Potential Tool

Schima superba (Theaceae) is a subtropical evergreen tree and is used widely for forest firebreaks and gardening. It is a plant that tolerates salt and typically accumulates elevated amounts of manganese in the leaves. With large ecological amplitude, this tree species grows quickly. Due to its substantial biomass, it has a great potential for soil remediation. To evaluate the thorough framework of the mRNA, we employed PacBio sequencing technology for the first time to generate S. Superba transcriptome. In this analysis, overall, 511,759 full length non-chimeric reads were acquired, and 163,834 high-quality full-length reads were obtained. Overall, 93,362 open reading frames were obtained, of which 78,255 were complete. In gene annotation analyses, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Non-Redundant (Nr) databases were allocated 91,082, 71,839, 38,914, and 38,376 transcripts, respectively. To identify long non-coding RNAs (lncRNAs), we utilized four computational methods associated with protein families (Pfam), Cooperative Data Classification (CPC), Coding Assessing Potential Tool (CPAT), and Coding Non-Coding Index (CNCI) databases and observed 8,551, 9,174, 20,720, and 18,669 lncRNAs, respectively. Moreover, nine genes were randomly selected for the expression analysis, which showed the highest expression of Gene 6 (Na_Ca_ex gene), and CAX (CAX-interacting protein 4) was higher in manganese (Mn)-treated group. This work provided significant number of full-length transcripts and refined the annotation of the reference genome, which will ease advanced genetic analyses of S. superba.

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Characterization of the Dynamic Transcriptome of a Herpesvirus with Long-read Single Molecule Real-Time Sequencing

Scientific Reports ◽

10.1038/srep43751 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 31

Author(s):

Dóra Tombácz ◽

Zsolt Balázs ◽

Zsolt Csabai ◽

Norbert Moldován ◽

Attila Szűcs ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Long Read

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

International Journal of Molecular Sciences ◽

10.3390/ijms20246350 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6350 ◽

Cited By ~ 2

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.

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Full-length transcriptome sequences of Agropyron cristatum facilitate the prediction of putative genes for thousand-grain weight in a wheat-A. cristatum translocation line

BMC Genomics ◽

10.1186/s12864-019-6416-4 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Shenghui Zhou ◽

Jinpeng Zhang ◽

Haiming Han ◽

Jing Zhang ◽

Huihui Ma ◽

...

Keyword(s):

Single Molecule ◽

Sequence Data ◽

Full Length ◽

Grain Weight ◽

Agropyron Cristatum ◽

Reference Sequence ◽

Translocation Line ◽

Thousand Grain Weight ◽

Sequencing Platform ◽

Transcriptome Sequences

Abstract Background Agropyron cristatum (L.) Gaertn. (2n = 4x = 28; genomes PPPP) is a wild relative of common wheat (Triticum aestivum L.) and provides many desirable genetic resources for wheat improvement. However, there is still a lack of reference genome and transcriptome information for A. cristatum, which severely impedes functional and molecular breeding studies. Results Single-molecule long-read sequencing technology from Pacific Biosciences (PacBio) was used to sequence full-length cDNA from a mixture of leaves, roots, stems and caryopses and constructed the first full-length transcriptome dataset of A. cristatum, which comprised 44,372 transcripts. As expected, the PacBio transcripts were generally longer and more complete than the transcripts assembled via the Illumina sequencing platform in previous studies. By analyzing RNA-Seq data, we identified tissue-enriched transcripts and assessed their GO term enrichment; the results indicated that tissue-enriched transcripts were enriched for particular molecular functions that varied by tissue. We identified 3398 novel and 1352 A. cristatum-specific transcripts compared with the wheat gene model set. To better apply this A. cristatum transcriptome, the A. cristatum transcripts were integrated with the wheat genome as a reference sequence to try to identify candidate A. cristatum transcripts associated with thousand-grain weight in a wheat-A. cristatum translocation line, Pubing 3035. Conclusions Full-length transcriptome sequences were used in our study. The present study not only provides comprehensive transcriptomic insights and information for A. cristatum but also proposes a new method for exploring the functional genes of wheat relatives under a wheat genetic background. The sequence data have been deposited in the NCBI under BioProject accession number PRJNA534411.

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

Forests ◽

10.3390/f11080866 ◽

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.

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Variant Phasing and Haplotypic Expression from Single-molecule Long-read Sequencing in Maize

10.1101/654533 ◽

2019 ◽

Cited By ~ 2

Author(s):

Bo Wang ◽

Elizabeth Tseng ◽

Primo Baybayan ◽

Kevin Eng ◽

Michael Regulski ◽

...

Keyword(s):

Single Molecule ◽

Genomic Analysis ◽

Full Length ◽

Specific Gene ◽

Population Genetic Analysis ◽

Functional Genomic Analysis ◽

Long Read ◽

Allele Specific ◽

Regulatory Effects ◽

Hybrid Lines

AbstractHaplotype phasing of genetic variants in maize is important for interpretation of the genome, population genetic analysis and functional genomic analysis of allelic activity. Accordingly, accurate methods for phasing the full-length isoforms are essential for functional genomics studies. We performed an isoform-level phasing study in maize, using two inbred lines and their reciprocal crosses, based on the single-molecule full-length cDNA sequencing. To phase and analyze the full-length transcripts between hybrids and parents, we developed a tool called IsoPhase. Using this tool, we validated the majority of SNPs called against matching short-read data and identified cases of allele-specific, gene-level and isoform-level expression. Our results revealed that maize parental lines and hybrid lines exhibit different splicing activities. After phasing 6,907 genes in two reciprocal hybrids using embryo, endosperm and root tissues, we annotated the SNPs and identified large-effect genes. In addition, based on single-molecule sequencing, we identified parent-of-origin isoforms in maize hybrids, distinct novel isoforms in maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase accuracy in studies of allelic expression.

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Full-length Transcriptome Analysis of Pecan (Carya illinoinensis) Kernels

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab182 ◽

2021 ◽

Author(s):

Chengcai Zhang ◽

Huadong Ren ◽

Xiaohua Yao ◽

Kailiang Wang ◽

Jun Chang

Keyword(s):

Single Molecule ◽

Molecular Mechanisms ◽

Draft Genome ◽

Alternative Polyadenylation ◽

Full Length ◽

Functional Annotations ◽

Predominant Type ◽

Long Read ◽

Non Coding Rnas ◽

Novel Isoforms

Abstract Pecan is rich in bioactive components such as fatty acids and flavonoids and is an important nut type worldwide. Therefore, the molecular mechanisms of phytochemical biosynthesis in pecan are a focus of research. Recently, a draft genome and several transcriptomes have been published. However, the full-length mRNA transcripts remain unclear, and the regulatory mechanisms behind the quality components biosynthesis and accumulation have not been fully investigated. In this study, single-molecule long read sequencing technology was used to obtain full-length transcripts of pecan kernels. In total, 37 504 isoforms of 16 702 genes were mapped to the reference genome. The numbers of known isoforms, new isoforms, and novel isoforms were 9013 (24.03%), 26 080 (69.54%), and 2411 (6.51%), respectively. Over 80% of the transcripts (30 751, 81.99%) had functional annotations. A total of 15 465 alternative splicing (AS) events and 65 761 alternative polyadenylation events were detected; wherein, the retained intron was the predominant type (5652, 36.55%) of AS. Furthermore, 1894 long non-coding RNAs and 1643 transcription factors were predicted using bioinformatics methods. Finally, the structural genes associated with fatty acid (FA) and flavonoid biosynthesis were characterized. A high frequency of AS accuracy (70.31%) was observed in FA synthesis-associated genes. The present study provides a full-length transcriptome dataset of pecan kernels, which will significantly enhance the understanding of the regulatory basis of phytochemical biosynthesis during pecan kernel maturation.

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SQANTI: extensive characterization of long read transcript sequences for quality control in full-length transcriptome identification and quantification

10.1101/118083 ◽

2017 ◽

Cited By ~ 9

Author(s):

Manuel Tardaguila ◽

Lorena de la Fuente ◽

Cristina Marti ◽

Cécile Pereira ◽

Francisco Jose Pardo-Palacios ◽

...

Keyword(s):

High Throughput Sequencing ◽

Full Length ◽

The Novel ◽

Extensive Evaluation ◽

Long Reads ◽

Long Read ◽

Novel Transcripts ◽

Mouse Transcriptome

ABSTRACTHigh-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in very well annotated organisms as mice and humans. Nonetheless, there is a need for studies and tools that characterize these novel isoforms. Here we present SQANTI, an automated pipeline for the classification of long-read transcripts that computes 47 descriptors that can be used to assess the quality of the data and of the preprocessing pipelines. We applied SQANTI to a neuronal mouse transcriptome using PacBio long reads and illustrate how the tool is effective in readily describing the composition of and characterizing the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach, and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, result more frequently in novel ORFs than novel UTRs and are enriched in both general metabolic and neural specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases we find that alternative isoforms are elusive to proteogenomics detection and are variable in protein changes with respect to the principal isoform of their genes. SQANTI allows the user to maximize the analytical outcome of long read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes. SQANTI is available at https://bitbucket.org/ConesaLab/sqanti.

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Comprehensive characterization of single cell full-length isoforms in human and mouse with long-read sequencing

10.1101/2020.08.10.243543 ◽

2020 ◽

Author(s):

Luyi Tian ◽

Jafar S. Jabbari ◽

Rachel Thijssen ◽

Quentin Gouil ◽

Shanika L. Amarasinghe ◽

...

Keyword(s):

Data Integration ◽

Single Cell ◽

Ribosome Biogenesis ◽

Single Cells ◽

Transcript Level ◽

Full Length ◽

Alternative Transcript ◽

Long Read ◽

Comprehensive Characterization

AbstractAlternative splicing shapes the phenotype of cells in development and disease. Long-read RNA-sequencing recovers full-length transcripts but has limited throughput at the single-cell level. Here we developed single-cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to overcome these issues and perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES, we performed the first comprehensive characterization of the full-length isoform landscape in single cells of different types and species and identified thousands of unannotated isoforms. We found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. Analysis at the transcript-level allowed data integration with scATAC-seq on individual promoters, improved correlation with protein expression data and linked mutations known to confer drug resistance to transcriptome heterogeneity. Our methods reveal previously unseen isoform complexity and provide a better framework for multi-omics data integration.

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