scholarly journals Combination of Single-Molecule Long-Read Sequencing and Illumina Sequencing Revealed the Mechanism of Anthocyanins Accumulation in an Ornamental Grass, Pennisetum Setaceum ‘Rubrum’

2021 ◽  
Author(s):  
Lingyun Liu ◽  
Ke Teng ◽  
Xifeng Fan ◽  
Hui Zhang ◽  
Chao Han ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Color Change ◽  
Enrichment Analysis ◽  
Underlying Mechanism ◽  
Full Length ◽  
Anthocyanin Accumulation ◽  
Sequencing Technology ◽  
Long Read ◽  
Pennisetum Setaceum

Abstract Pennisetum setaceum ‘Rubrum’ is an ornamental herb with purple leaves, and it is widely used in the construction of landscaping. However, the current next generation sequencing (NGS) transcriptome information is not satisfactory mainly because of the enormous difficulty in obtaining full-length transcripts. What’s more, the molecular mechanisms of anthocyanin accumulation have not been thoroughly studied. In this study, we used PacBio full-length transcriptome sequencing combined with NGS sequencing technology to conduct transcriptome analysis on leaves showing different colors at different stages to clarify the molecular mechanism involved in the color change of P. setaceum ‘Rubrum’. A total of 280,413 full-length non-chimeric reads (FLNC) sequences were obtained based on single-molecule long-read sequencing technology. We obtained 140,633 high quality (HQ) transcripts and 2,683 low quality (LQ) transcripts and identified 5,352 alternative splicing (AS). In addition, a total of 93,066 ORFs, including 57,457 full open links and 2,910 lncRNA sequences were screened out. Furthermore, a total of 10,795 differentially expressed genes were identified. Gene ontology (GO) cluster and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the underlying mechanism of anthocyanin accumulation. In this study, to our best knowledge, we provided the full-length transcriptome information of P. setaceum ‘Rubrum’ for the first time. The underlying mechanism of anthocyanin accumulation in P. setaceum ‘Rubrum’ was further discussed based on the newly generated transcriptome data. The information will not only facilitate the gene function studies but also pave the way for future breeding projects of Pennisetum setaceum .

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

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.

scholarly journals Full-Length Transcriptome Analysis Reveals Candidate Genes Involved in Terpenoid Biosynthesis in Artemisia argyi

2021 ◽  
Vol 12 ◽  
Author(s):  
Yupeng Cui ◽  
Xinqiang Gao ◽  
Jianshe Wang ◽  
Zengzhen Shang ◽  
Zhibin Zhang ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Enrichment Analysis ◽  
Full Length ◽  
Functional Enrichment ◽  
Terpenoid Biosynthesis ◽  
Sequencing Data ◽  
Artemisia Argyi

Artemisia argyi is an important medicinal plant widely utilized for moxibustion heat therapy in China. The terpenoid biosynthesis process in A. argyi is speculated to play a key role in conferring its medicinal value. However, the molecular mechanism underlying terpenoid biosynthesis remains unclear, in part because the reference genome of A. argyi is unavailable. Moreover, the full-length transcriptome of A. argyi has not yet been sequenced. Therefore, in this study, de novo transcriptome sequencing of A. argyi's root, stem, and leaf tissues was performed to obtain those candidate genes related to terpenoid biosynthesis, by combining the PacBio single-molecule real-time (SMRT) and Illumina sequencing NGS platforms. And more than 55.4 Gb of sequencing data and 108,846 full-length reads (non-chimeric) were generated by the Illumina and PacBio platform, respectively. Then, 53,043 consensus isoforms were clustered and used to represent 36,820 non-redundant transcripts, of which 34,839 (94.62%) were annotated in public databases. In the comparison sets of leaves vs roots, and leaves vs stems, 13,850 (7,566 up-regulated, 6,284 down-regulated) and 9,502 (5,284 up-regulated, 4,218 down-regulated) differentially expressed transcripts (DETs) were obtained, respectively. Specifically, the expression profile and KEGG functional enrichment analysis of these DETs indicated that they were significantly enriched in the biosynthesis of amino acids, carotenoids, diterpenoids and flavonoids, as well as the metabolism processes of glycine, serine and threonine. Moreover, multiple genes encoding significant enzymes or transcription factors related to diterpenoid biosynthesis were highly expressed in the A. argyi leaves. Additionally, several transcription factor families, such as RLK-Pelle_LRR-L-1 and RLK-Pelle_DLSV, were also identified. In conclusion, this study offers a valuable resource for transcriptome information, and provides a functional genomic foundation for further research on molecular mechanisms underlying the medicinal use of A. argyi leaves.

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

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.

scholarly journals Allosteric activation of preformed EGF receptor dimers by binding of a single ligand

2021 ◽  
Author(s):  
Endang Purba ◽  
Ei-ichiro Saita ◽  
Reetesh Akhouri ◽  
Lars-Göran Öfverstedt ◽  
Gunnar Wilken ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Electron Tomography ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Cancer Therapies ◽  
Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after activation. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilised by ligand binding. Consistently, optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

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 A Full-Length Reference Floral Transcriptome of Boehmeria tricuspis Provides Insights into Apomeiosis and Polyploidy

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Qing Tang ◽  
Ying Xu ◽  
Canhui Deng ◽  
Chaohua Cheng ◽  
Zhigang Dai ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Reproductive Mode ◽  
Full Length ◽  
Ploidy Levels ◽  
Reproductive Genes ◽  
Stress Response Genes ◽  
Long Read ◽  
Floral Transcriptome ◽  
Female Flowers ◽  
Significant Factors

Boehmeria tricuspis (Hance) Makino constitutes a hardy herbaceous or shrubby perennial native to East Asia that includes different ploidy levels and reproductive modes (diplosporous to sexual). Although several apomeiosis-associated genes have been described, the genetic control and molecular mechanisms underlying apomeiosis remain poorly understood. Moreover, the basis of the correlation between polyploidy and apomixis has not yet been clarified. We utilized long-read sequencing to produce a full-length reference floral transcriptome of B. tricuspis. Based on the generated database, gene expression of the female flowers of different ploidy levels and reproductive mode cytotypes was compared. Overall, 1,387 genes related to apomeiosis, 217 genes related to ploidy, and 9 genes associated with both apomixis and ploidy were identified. Gene Ontology analyses of this set of transcripts indicated reproductive genes, especially those related to “cell differentiation” and “cell cycle process,” as significant factors regulating apomeiosis. Furthermore, our results suggested that different expressions of stress response genes might be important in the preparation for apomeiosis transition. In addition, our observations indicated that the expression of apomeiosis may not depend on polyploidy but rather on deregulation of the sexual pathway in B. tricuspis.

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 Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens

2019 ◽  
Vol 47 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Yann Fichou ◽  
Isabelle Berlivet ◽  
Gaëlle Richard ◽  
Christophe Tournamille ◽  
Lilian Castilho ◽  
...  
Keyword(s):  
Blood Group ◽  
Single Molecule ◽  
Pcr Amplification ◽  
Null Alleles ◽  
Sequencing Technology ◽  
Gene Encoding ◽  
Next Generation Sequencing Technology ◽  
Sequencing Technologies ◽  
Long Read ◽  
Long Range Pcr

Background: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a “longitudinal” overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

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

2019 ◽  
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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