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Asset Tag: RNA-seq

April 21, 2020

Analysis of Transcriptome and Epitranscriptome in Plants Using PacBio Iso-Seq and Nanopore-Based Direct RNA Sequencing.

Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses.

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April 21, 2020

Transcriptome analysis based on a combination of sequencing platforms provides insights into leaf pigmentation in Acer rubrum.

Red maple (Acer rubrum L.) is one of the most common and widespread trees with colorful leaves. We found a mutant with red, yellow, and green leaf phenotypes in different branches, which provided ideal materials with the same genetic relationship, and little interference from the environment, for the study of complex metabolic networks that underly variations in the coloration of leaves. We applied a combination of NGS and SMRT sequencing to various red maple tissues.A total of 125,448 unigenes were obtained, of which 46 and 69 were thought to be related to the synthesis of anthocyanins and carotenoids, respectively. In addition, 88 unigenes were presumed to be involved in the chlorophyll metabolic pathway. Based on a comprehensive analysis of the pigment gene expression network, the mechanisms of leaf color were investigated. The massive accumulation of Cy led to its higher content and proportion than other pigments, which caused the redness of leaves. Yellow coloration was the result of the complete decomposition of chlorophyll pigments, the unmasking of carotenoid pigments, and a slight accumulation of Cy.This study provides a systematic analysis of color variations in the red maple. Moreover, mass sequence data obtained by deep sequencing will provide references for the controlled breeding of red maple.

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April 21, 2020

Iso-Seq analysis of the Taxus cuspidata transcriptome reveals the complexity of Taxol biosynthesis.

Taxus cuspidata is well known worldwide for its ability to produce Taxol, one of the top-selling natural anticancer drugs. However, current Taxol production cannot match the increasing needs of the market, and novel strategies should be considered to increase the supply of Taxol. Since the biosynthetic mechanism of Taxol remains largely unknown, elucidating this pathway in detail will be very helpful in exploring alternative methods for Taxol production.Here, we sequenced Taxus cuspidata transcriptomes with next-generation sequencing (NGS) and third-generation sequencing (TGS) platforms. After correction with Illumina reads and removal of redundant reads, more than 180,000 nonredundant transcripts were generated from the raw Iso-Seq data. Using Cogent software and an alignment-based method, we identified a total of 139 cytochrome P450s (CYP450s), 31 BAHD acyltransferases (ACTs) and 1940 transcription factors (TFs). Based on phylogenetic and coexpression analysis, we identified 9 CYP450s and 7 BAHD ACTs as potential lead candidates for Taxol biosynthesis and 6 TFs that are possibly involved in the regulation of this process. Using coexpression analysis of genes known to be involved in Taxol biosynthesis, we elucidated the stem biosynthetic pathway. In addition, we analyzed the expression patterns of 12 characterized genes in the Taxol pathway and speculated that the isoprene precursors for Taxol biosynthesis were mainly synthesized via the MEP pathway. In addition, we found and confirmed that the alternative splicing patterns of some genes varied in different tissues, which may be an important tissue-specific method of posttranscriptional regulation.A strategy was developed to generate corrected full-length or nearly full-length transcripts without assembly to ensure sequence accuracy, thus greatly improving the reliability of coexpression and phylogenetic analysis and greatly facilitating gene cloning and characterization. This strategy was successfully utilized to elucidate the Taxol biosynthetic pathway, which will greatly contribute to the goals of improving the Taxol content in Taxus spp. using molecular breeding or plant management strategies and synthesizing Taxol in microorganisms using synthetic biological technology.

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April 21, 2020

De novo transcriptome assembly of the cubomedusa Tripedalia cystophora, including the analysis of a set of genes involved in peptidergic neurotransmission.

The phyla Cnidaria, Placozoa, Ctenophora, and Porifera emerged before the split of proto- and deuterostome animals, about 600 million years ago. These early metazoans are interesting, because they can give us important information on the evolution of various tissues and organs, such as eyes and the nervous system. Generally, cnidarians have simple nervous systems, which use neuropeptides for their neurotransmission, but some cnidarian medusae belonging to the class Cubozoa (box jellyfishes) have advanced image-forming eyes, probably associated with a complex innervation. Here, we describe a new transcriptome database from the cubomedusa Tripedalia cystophora.Based on the combined use of the Illumina and PacBio sequencing technologies, we produced a highly contiguous transcriptome database from T. cystophora. We then developed a software program to discover neuropeptide preprohormones in this database. This script enabled us to annotate seven novel T. cystophora neuropeptide preprohormone cDNAs: One coding for 19 copies of a peptide with the structure pQWLRGRFamide; one coding for six copies of a different RFamide peptide; one coding for six copies of pQPPGVWamide; one coding for eight different neuropeptide copies with the C-terminal LWamide sequence; one coding for thirteen copies of a peptide with the RPRAamide C-terminus; one coding for four copies of a peptide with the C-terminal GRYamide sequence; and one coding for seven copies of a cyclic peptide, of which the most frequent one has the sequence CTGQMCWFRamide. We could also identify orthologs of these seven preprohormones in the cubozoans Alatina alata, Carybdea xaymacana, Chironex fleckeri, and Chiropsalmus quadrumanus. Furthermore, using TBLASTN screening, we could annotate four bursicon-like glycoprotein hormone subunits, five opsins, and 52 other family-A G protein-coupled receptors (GPCRs), which also included two leucine-rich repeats containing G protein-coupled receptors (LGRs) in T. cystophora. The two LGRs are potential receptors for the glycoprotein hormones, while the other GPCRs are candidate receptors for the above-mentioned neuropeptides.By combining Illumina and PacBio sequencing technologies, we have produced a new high-quality de novo transcriptome assembly from T. cystophora that should be a valuable resource for identifying the neuronal components that are involved in vision and other behaviors in cubomedusae.

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April 21, 2020

Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei.

In Trypanosoma brucei, most mitochondrial mRNAs undergo editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for translation. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes mRNA degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point likely prevents translation of incompletely edited mRNAs. We also find that RNA editing substrate binding complex (RESC) mediates the interaction between the 5′ end-bound pyrophosphohydrolase MERS1 and 3′ end-associated KPAF4 to enable mRNA circularization. This event appears to be critical for edited mRNA stability.

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April 21, 2020

Comparative transcriptome analysis identified candidate genes involved in mycelium browning in Lentinula edodes.

Lentinula edodes is one of the most popular edible mushroom species in the world and contains useful medicinal components, such as lentinan. The light-induced formation of brown film on the vegetative mycelial tissues of L. edodes is an important process for ensuring the quantity and quality of this edible mushroom. To understand the molecular mechanisms underlying this critical developmental process in L. edodes, we characterized the morphological phenotypic changes in a strain, Chamaram, associated with abnormal brown film formation and compared its genome-wide transcriptional features.In the present study, we performed genome-wide transcriptome analyses of different vegetative mycelium growth phenotypes, namely, early white, normal brown, and defective dark yellow partial brown films phenotypes which were exposed to different light conditions. The analysis revealed the identification of clusters of genes specific to the light-induced brown film phenotypes. These genes were significantly associated with light sensing via photoreceptors such as FMN- and FAD-bindings, signal transduction by kinases and GPCRs, melanogenesis via activation of tyrosinases, and cell wall degradation by glucanases, chitinases, and laccases, which suggests these processes are involved in the formation of mycelial browning in L. edodes. Interestingly, hydrophobin genes such as SC1 and SC3 exhibited divergent expression levels in the normal and abnormal brown mycelial films, indicating the ability of these genes to act in fruiting body initiation and formation of dikaryotic mycelia. Furthermore, we identified the up-regulation of glycoside hydrolase domain-containing genes in the normal brown film but not in the abnormal film phenotype, suggesting that cell wall degradation in the normal brown film phenotype is crucial in the developmental processes related to the initiation and formation of fruiting bodies.This study systematically analysed the expression patterns of light-induced browning-related genes in L. edodes. Our findings provide information for further investigations of browning formation mechanisms in L. edodes and a foundation for future L. edodes breeding.

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April 21, 2020

The genome of the soybean cyst nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes.

Heterodera glycines, commonly referred to as the soybean cyst nematode (SCN), is an obligatory and sedentary plant parasite that causes over a billion-dollar yield loss to soybean production annually. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. The parasitic success of H. glycines relies on the comprehensive re-engineering of an infection site into a syncytium, as well as the long-term suppression of host defense to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms of effector acquisition, diversification, and selection need to be understood before effective control strategies can be developed, but the lack of an annotated genome has been a major roadblock.Here, we use PacBio long-read technology to assemble a H. glycines genome of 738 contigs into 123?Mb with annotations for 29,769 genes. The genome contains significant numbers of repeats (34%), tandem duplicates (18.7?Mb), and horizontal gene transfer events (151 genes). A large number of putative effectors (431 genes) were identified in the genome, many of which were found in transposons.This advance provides a glimpse into the host and parasite interplay by revealing a diversity of mechanisms that give rise to virulence genes in the soybean cyst nematode, including: tandem duplications containing over a fifth of the total gene count, virulence genes hitchhiking in transposons, and 107 horizontal gene transfers not reported in other plant parasitic nematodes thus far. Through extensive characterization of the H. glycines genome, we provide new insights into H. glycines biology and shed light onto the mystery underlying complex host-parasite interactions. This genome sequence is an important prerequisite to enable work towards generating new resistance or control measures against H. glycines.

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April 21, 2020

Characterization of a male specific region containing a candidate sex determining gene in Atlantic cod.

The genetic mechanisms determining sex in teleost fishes are highly variable and the master sex determining gene has only been identified in few species. Here we characterize a male-specific region of 9?kb on linkage group 11 in Atlantic cod (Gadus morhua) harboring a single gene named zkY for zinc knuckle on the Y chromosome. Diagnostic PCR test of phenotypically sexed males and females confirm the sex-specific nature of the Y-sequence. We identified twelve highly similar autosomal gene copies of zkY, of which eight code for proteins containing the zinc knuckle motif. 3D modeling suggests that the amino acid changes observed in six copies might influence the putative RNA-binding specificity. Cod zkY and the autosomal proteins zk1 and zk2 possess an identical zinc knuckle structure, but only the Y-specific gene zkY was expressed at high levels in the developing larvae before the onset of sex differentiation. Collectively these data suggest zkY as a candidate master masculinization gene in Atlantic cod. PCR amplification of Y-sequences in Arctic cod (Arctogadus glacialis) and Greenland cod (Gadus macrocephalus ogac) suggests that the male-specific region emerged in codfishes more than 7.5 million years ago.

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April 21, 2020

Using Pan RNA-Seq Analysis to Reveal the Ubiquitous Existence of 5′ and 3′ End Small RNAs.

In this study, we used pan RNA-seq analysis to reveal the ubiquitous existence of both 5′ and 3′ end small RNAs (5′ and 3′ sRNAs). 5′ and 3′ sRNAs alone can be used to annotate nuclear non-coding and mitochondrial genes at 1-bp resolution and identify new steady RNAs, which are usually transcribed from functional genes. Then, we provided a simple and cost effective way for the annotation of nuclear non-coding and mitochondrial genes and the identification of new steady RNAs, particularly long non-coding RNAs (lncRNAs). Using 5′ and 3′ sRNAs, the annotation of human mitochondrial was corrected and a novel ncRNA named non-coding mitochondrial RNA 1 (ncMT1) was reported for the first time in this study. We also found that most of human tRNA genes have downstream lncRNA genes as lncTRS-TGA1-1 and corrected the misunderstanding of them in previous studies. Using 5′, 3′, and intronic sRNAs, we reported for the first time that enzymatic double-stranded RNA (dsRNA) cleavage and RNA interference (RNAi) might be involved in the RNA degradation and gene expression regulation of U1 snRNA in human. We provided a different perspective on the regulation of gene expression in U1 snRNA. We also provided a novel view on cancer and virus-induced diseases, leading to find diagnostics or therapy targets from the ribonuclease III (RNase III) family and its related pathways. Our findings pave the way toward a rediscovery of dsRNA cleavage and RNAi, challenging classical theories.

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April 21, 2020

The tech for the next decade: promises and challenges in genome biology.

The 19th Annual Advances in Genome Biology and Technology (AGBT) meeting came back to Marco Island, Florida, and was held in the renovated venue from 27 February to 2 March 2019. The meeting showed a variety of new technology, both in wet lab and in bioinformatics. This year’s themes included single-cell technology and applications, spatially resolved gene expression measurements, new sequencing platforms, genome assembly and variation, and long and linked reads.

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April 21, 2020

Transcriptomic profiles of 33 opium poppy samples in different tissues, growth phases, and cultivars.

Opium poppy is one of the most important medicinal plants and remains the only commercial resource of morphinan-based painkillers. However, little is known about the regulatory mechanisms involved in benzylisoquinoline alkaloids (BIAs) biosynthesis in opium poppy. Herein, the full-length transcriptome dataset of opium poppy was constructed for the first time in accompanied with the 33 samples of Illumina transcriptome data from different tissues, growth phases and cultivars. The long-read sequencing produced 902,140 raw reads with 55,114 high-quality transcripts, and short-read sequencing produced 1,923,679,864 clean reads with an average Q30 rate of 93%. The high-quality transcripts were subsequently quantified using the short reads, and the expression of each unigene among different samples was calculated as reads per kilobase per million mapped reads (RPKM). These data provide a foundation for opium poppy transcriptomic analysis, which may aid in capturing splice variants and some non-coding RNAs involved in the regulation of BIAs biosynthesis. It can also be used for genome assembly and annotation which will favor in new transcript identification.

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April 21, 2020

Identification of Candidate Genes for the Plateau Adaptation of a Tibetan Amphipod, Gammarus lacustris, Through Integration of Genome and Transcriptome Sequencing.

The amphipod Gammarus lacustris has been distributing in the Tibetan region with well-known uplifts of the Tibetan plateau. It is hence considered as a good model for investigating stress adaptations of the plateau. Here, we sequenced the whole-genome and full-length transcriptome of G. lacustris, and compared the transcriptome results with its counterpart Gammarus pisinnus from a nearby plain. Our main goal was to provide a genomic resource for investigation of genetic mechanisms, by which G. lacustris adapted to living on the plateau. The final draft genome assembly of G. lacustris was 5.07 gigabases (Gb), and it contained 443,304 scaffolds (>2 kb) with an N50 of 2,578 bp. A total of 8,858 unigenes were predicted in the full-length transcriptome of G. lacustris, with an average gene length of 1,811 bp. Compared with the G. pisinnus transcriptome, 2,672 differentially expressed genes (DEGs) were up-regulated and 2,881 DEGs were down-regulated in the G. lacustris transcriptome. Along with these critical DEGs, several enriched metabolic pathways, such as oxidative phosphorylation, ribosome, cell energy homeostasis, glycolysis and gluconeogenesis, were predicted to play essential roles in the plateau adaptation. In summary, the present study provides a genomic basis for understanding the plateau adaption of G. lacustris, which lays a fundamental basis for further biological and ecological studies on other resident aquatic species in the Tibetan plateau.

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April 21, 2020

The complexity of the Fragaria x ananassa (octoploid) transcriptome by single-molecule long-read sequencing.

Strawberry (Fragaria x ananassa) is an allopolyploid species with diverse and complex transcripts. The regulatory mechanisms of fruit development and maturation have been extensively studied; however, little is known about the signaling mechanisms that direct this process in octoploid strawberry (Fragaria x ananassa). Here, we used long-read sequencing (LRS) technology and RNA-seq analysis to investigate the diversity and complexity of the polyploid transcriptome and differentially expressed transcripts along four successive fruit developmental stages of cultivated strawberry. We obtained a reference transcriptome with 119,897 unique full-length isoforms, including 2017 new isoforms and 2510 long noncoding RNAs. Based on the genome of the plausible progenitor (Fragaria vesca), 20,229 alternative splicing (AS) events were identified. Using this transcriptome, we found 17,485 differentially expressed transcripts during strawberry fruit development, including 527 transcription factors (TFs) belonging to 41 families. The expression profiles of all members of the auxin, ABA pathway, and anthocyanin biosynthesis gene families were also examined, and many of them were highly expressed at the ripe fruit stage, strongly indicating that the role of those genes is in the regulation of fruit ripening. We produce a high-quality reference transcriptome for octoploid strawberry, including much of the full-length transcript diversity, to help understand the regulatory mechanisms of fruit development and maturation of polyploid species, particularly via elucidation of the biochemical pathways involved in auxin, ABA, and anthocyanin biosynthesis.

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April 21, 2020

Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.).

Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress.In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24?h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551?bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress.Overall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering.

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April 21, 2020

Evaluation of reference genes for normalizing RT-qPCR in leaves and suspension cells of Cephalotaxus hainanensis under various stimuli.

Reverse transcription quantitative real-time PCR (RT-qPCR) is a widely used approach for investigating gene expression levels in plants because of its high reproducibility, sensitivity, accuracy and rapidness. Evaluation of reference genes for normalizing RT-qPCR data is a necessary step, especially in new plant varieties. Cephalotaxus hainanensis is a precious medicinal plant belonging to the family of Cephalotaxaceae and no RT-qPCR studies have been reported on it.In this study, 9 candidate reference genes were selected from the transcriptome data of C. hainanensis; 3 statistical algorithms (geNorm, NormFinder, BestKeeper) were applied to evaluate their expression stabilities through 180 samples under 6 stimuli treatments in leaves and leaf-derived suspension cultured cells; a comprehensive stabilities ranking was also performed by RefFinder. The results showed that suitable reference genes in C. hainanensis should be selected for normalization relative to different experimental sets. 18S showed a higher stability than other candidate reference genes which ranked at the top two suitable genes under all experimental setups in this study.This study is the first to evaluate the stability of reference genes in C. hainanensis and supply an important foundation to use the RT-qPCR for an accurate and far-reaching gene expression analysis in C. hainanensis.

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