scholarly journals Integrative Transcriptome Sequencing Reveals the Molecular Difference of Maturation Process of Ovary and Testis in Mud Crab Scylla paramamosain

2021 ◽  
Vol 8 ◽  
Author(s):  
Wenxiao Cui ◽  
Qin Yang ◽  
Yin Zhang ◽  
Ardavan Farhadi ◽  
Huan Fang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gonadal Development ◽  
Scylla Paramamosain ◽  
Gonadal Differentiation ◽  
Rna Seq ◽  
Mud Crab ◽  
Nutritional Values ◽  
Long Read ◽  
Novel Transcripts ◽  
Polyadenylation Sites

The mud crab Scylla paramamosain is a species with significant sexual dimorphism in growth rate and body size, of which the females are of higher economic and nutritional values than the males. Accordingly, there is an urgent need to explore the molecular mechanism underlying sex determination and gonadal differentiation. The single-molecule long-read technology combining with RNA sequencing was employed to construct a full-length transcriptome for gonads of S. paramamosain. In total, 1,562,819 FLNC reads were obtained from 1,813,758 reads of inserts (ROIs). Among them, the 10,739 fusion isoforms corresponded to 23,634 reads and were involved in 5,369 genes in the reference annotation. According to the criteria for new transcripts, a total of 213,809 isoforms were recognized as novel transcripts and then matched against Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), NR, Swissprot, and KOG databases. We also identified 22,313 SSRs, 169,559 lncRNAs, and 25,451 SNPs. Additionally, 349,854 alternative splicing (AS) events from 8,430 gene models were detected, and 5,129 polyadenylation sites were profiled from 3,090 genes. GO and KEGG annotation indicated that AS and APA probably play important roles in the gonadal development and maturation. Besides, the DEGs associated with gonadal development and maturation were identified and analyzed based on the RNA-Seq data.

scholarly journals A long-read RNA-seq approach to identify novel transcripts of very large genes

2020 ◽  
Vol 30 (6) ◽  
pp. 885-897
Author(s):  
Prech Uapinyoying ◽  
Jeremy Goecks ◽  
Susan M. Knoblach ◽  
Karuna Panchapakesan ◽  
Carsten G. Bonnemann ◽  
...  
Keyword(s):  
Rna Seq ◽  
Long Read ◽  
Novel Transcripts ◽  
Large Genes

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 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 Benchmarking sequencing methods and tools that facilitate the study of alternative polyadenylation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Ankeeta Shah ◽  
Briana E. Mittleman ◽  
Yoav Gilad ◽  
Yang I. Li
Keyword(s):  
Single Molecule ◽  
Alternative Polyadenylation ◽  
Regulatory Elements ◽  
Polyadenylation Site ◽  
Rna Seq ◽  
Computational Tools ◽  
Protein Coding ◽  
Short Read ◽  
Processing Event ◽  
Polyadenylation Sites

Abstract Background Alternative cleavage and polyadenylation (APA), an RNA processing event, occurs in over 70% of human protein-coding genes. APA results in mRNA transcripts with distinct 3′ ends. Most APA occurs within 3′ UTRs, which harbor regulatory elements that can impact mRNA stability, translation, and localization. Results APA can be profiled using a number of established computational tools that infer polyadenylation sites from standard, short-read RNA-seq datasets. Here, we benchmarked a number of such tools—TAPAS, QAPA, DaPars2, GETUTR, and APATrap— against 3′-Seq, a specialized RNA-seq protocol that enriches for reads at the 3′ ends of genes, and Iso-Seq, a Pacific Biosciences (PacBio) single-molecule full-length RNA-seq method in their ability to identify polyadenylation sites and quantify polyadenylation site usage. We demonstrate that 3′-Seq and Iso-Seq are able to identify and quantify the usage of polyadenylation sites more reliably than computational tools that take short-read RNA-seq as input. However, we find that running one such tool, QAPA, with a set of polyadenylation site annotations derived from small quantities of 3′-Seq or Iso-Seq can reliably quantify variation in APA across conditions, such asacross genotypes, as demonstrated by the successful mapping of alternative polyadenylation quantitative trait loci (apaQTL). Conclusions We envisage that our analyses will shed light on the advantages of studying APA with more specialized sequencing protocols, such as 3′-Seq or Iso-Seq, and the limitations of studying APA with short-read RNA-seq. We provide a computational pipeline to aid in the identification of polyadenylation sites and quantification of polyadenylation site usages using Iso-Seq data as input.

scholarly journals Transcriptome innovations in primates revealed by single-molecule long-read sequencing

2021 ◽  
Author(s):  
Luis Ferrandez-Peral ◽  
Xiaoyu Zhan ◽  
Marina Alvarez-Estape ◽  
Cristina Chiva ◽  
Paula Esteller-Cucala ◽  
...  
Keyword(s):  
Single Molecule ◽  
Great Apes ◽  
Primate Evolution ◽  
Lymphoblastoid Cell Lines ◽  
Short Read ◽  
Limited Impact ◽  
Short Read Sequencing ◽  
Important Target ◽  
Long Read ◽  
Novel Transcripts

Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Our transcriptomes reveal thousands of novel transcripts, some of them under active translation, expanding and completing the repertoire of primate gene models. Our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.

scholarly journals PacBio and Illumina RNA Sequencing Identify Alternative Splicing Events in Response to Cold Stress in Two Poplar Species

2021 ◽  
Vol 12 ◽  
Author(s):  
Jingli Yang ◽  
Wanqiu Lv ◽  
Liying Shao ◽  
Yanrui Fu ◽  
Haimei Liu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Stress Response ◽  
Cold Stress ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Intron Retention ◽  
Global Analysis ◽  
Populus Trichocarpa ◽  
Rna Seq ◽  
Long Read

In eukaryotes, alternative splicing (AS) is a crucial regulatory mechanism that modulates mRNA diversity and stability. The contribution of AS to stress is known in many species related to stress, but the posttranscriptional mechanism in poplar under cold stress is still unclear. Recent studies have utilized the advantages of single molecular real-time (SMRT) sequencing technology from Pacific Bioscience (PacBio) to identify full-length transcripts. We, therefore, used a combination of single-molecule long-read sequencing and Illumina RNA sequencing (RNA-Seq) for a global analysis of AS in two poplar species (Populus trichocarpa and P. ussuriensis) under cold stress. We further identified 1,261 AS events in P. trichocarpa and 2,101 in P. ussuriensis among which intron retention, with a frequency of more than 30%, was the most prominent type under cold stress. RNA-Seq data analysis and annotation revealed the importance of calcium, abscisic acid, and reactive oxygen species signaling in cold stress response. Besides, the low temperature rapidly induced multiple splicing factors, transcription factors, and differentially expressed genes through AS. In P. ussuriensis, there was a rapid occurrence of AS events, which provided a new insight into the complexity and regulation of AS during cold stress response in different poplar species for the first time.

scholarly journals Long-read sequencing reveals genomic structural variations that underlie creation of quality protein maize

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Changsheng Li ◽  
Xiaoli Xiang ◽  
Yongcai Huang ◽  
Yong Zhou ◽  
Dong An ◽  
...  
Keyword(s):  
South African ◽  
Single Molecule ◽  
Potential Role ◽  
Quality Protein Maize ◽  
Rna Seq ◽  
Maize Endosperm ◽  
Structural Variations ◽  
Optical Map ◽  
Long Read ◽  
Endosperm Formation

AbstractMutation of o2 doubles maize endosperm lysine content, but it causes an inferior kernel phenotype. Developing quality protein maize (QPM) by introgressing o2 modifiers (Mo2s) into the o2 mutant benefits millions of people in developing countries where maize is a primary protein source. Here, we report genome sequence and annotation of a South African QPM line K0326Y, which is assembled from single-molecule, real-time shotgun sequencing reads collinear with an optical map. We achieve a N50 contig length of 7.7 million bases (Mb) directly from long-read assembly, compared to those of 1.04 Mb for B73 and 1.48 Mb for Mo17. To characterize Mo2s, we map QTLs to chromosomes 1, 6, 7, and 9 using an F2 population derived from crossing K0326Y and W64Ao2. RNA-seq analysis of QPM and o2 endosperms reveals a group of differentially expressed genes that coincide with Mo2 QTLs, suggesting a potential role in vitreous endosperm formation.

scholarly journals Characterization and analysis of the transcriptome in Gymnocypris selincuoensis on the Qinghai-Tibetan Plateau using single-molecule long-read sequencing and RNA-seq

DNA Research ◽  
2019 ◽  
Vol 26 (4) ◽  
pp. 353-363 ◽  
Author(s):  
Xiu Feng ◽  
Yintao Jia ◽  
Ren Zhu ◽  
Kang Chen ◽  
Yifeng Chen
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Genomic Analysis ◽  
Open Reading Frames ◽  
Tibet Plateau ◽  
Comparative Genomic ◽  
Rna Seq ◽  
Gene Expression Atlas ◽  
Expression Atlas ◽  
Long Read

Abstract The lakes on the Qinghai-Tibet Plateau (QTP) are the largest and highest lake group in the world. Gymnocypris selincuoensis is the only cyprinid fish living in lake Selincuo, the largest lake on QTP. However, its genetic resource is still blank, limiting studies on molecular and genetic analysis. In this study, the transcriptome of G. selincuoensis was first generated by using PacBio Iso-Seq and Illumina RNA-seq. A full-length (FL) transcriptome with 75,435 transcripts was obtained by Iso-Seq with N50 length of 3,870 bp. Among all transcripts, 75,016 were annotated to public databases, 64,710 contain complete open reading frames and 2,811 were long non-coding RNAs. Based on all- vs.-all BLAST, 2,069 alternative splicing events were detected, and 80% of them were validated by reverse transcription polymerase chain reaction (RT-PCR). Tissue gene expression atlas showed that the number of detected expressed transcripts ranged from 37,397 in brain to 19,914 in muscle, with 10,488 transcripts detected in all seven tissues. Comparative genomic analysis with other cyprinid fishes identified 77 orthologous genes with potential positive selection (Ka/Ks > 0.3). A total of 56,696 perfect simple sequence repeats were identified from FL transcripts. Our results provide valuable genetic resources for further studies on adaptive evolution, gene expression and population genetics in G. selincuoensis and other congeneric fishes.

scholarly journals Transcriptome assembly from long-read RNA-seq alignments with StringTie2

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Sam Kovaka ◽  
Aleksey V. Zimin ◽  
Geo M. Pertea ◽  
Roham Razaghi ◽  
Steven L. Salzberg ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcriptome Assembly ◽  
Rna Seq ◽  
Ability To Work ◽  
Single Molecule Sequencing ◽  
Short Read ◽  
New Methods ◽  
Long Reads ◽  
Long Read

AbstractRNA sequencing using the latest single-molecule sequencing instruments produces reads that are thousands of nucleotides long. The ability to assemble these long reads can greatly improve the sensitivity of long-read analyses. Here we present StringTie2, a reference-guided transcriptome assembler that works with both short and long reads. StringTie2 includes new methods to handle the high error rate of long reads and offers the ability to work with full-length super-reads assembled from short reads, which further improves the quality of short-read assemblies. StringTie2 is more accurate and faster and uses less memory than all comparable short-read and long-read analysis tools.

scholarly journals Long read, isoform aware sequencing of mouse nucleus accumbens after chronic cocaine treatment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Molly Estill ◽  
Efrain Ribeiro ◽  
Nancy J. Francoeur ◽  
Melissa L. Smith ◽  
Robert Sebra ◽  
...  
Keyword(s):  
Nucleus Accumbens ◽  
Single Molecule ◽  
Sequencing Analysis ◽  
The Novel ◽  
Polyadenylated Rna ◽  
Chronic Cocaine ◽  
Long Read ◽  
Novel Transcripts ◽  
Brain Reward ◽  
Comprehensive Reference

AbstractTo better understand the full-length transcriptome of the nucleus accumbens (NAc)—a key brain reward region—in chronic cocaine treatment, we perform the first single molecule, long-read sequencing analysis using the Iso-seq method to detect 42,114 unique transcripts from mouse NAc polyadenylated RNA. Using GENCODE annotation as a reference, we find that over half of the Iso-seq derived transcripts are annotated, while 46% of them harbor novel splicing events in known genes; around 1% of them correspond to other types of novel transcripts, such as fusion, antisense and intergenic. Approximately 34% of the novel transcripts are matched with a compiled transcriptome assembled from published short-read data from various tissues, with the remaining 69% being unique to NAc. These data provide a more complete picture of the NAc transcriptome than existing annotations and can serve as a comprehensive reference for future transcriptomic analyses of this important brain reward region.

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