Non-redundant Full-length Transcripts Research Articles

Single-Molecule Real-Time Sequencing for Identifying Sexual-Dimorphism-Related Transcriptomes and Genes in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

The Chinese soft-shelled turtle (Pelodiscus sinensis), an economically important aquatic species in China, displays considerable sexual dimorphism: the male P. sinensis is larger and, thus, more popular in the market. In this study, we obtained the full-length (FL) transcriptome data of P. sinensis by using Pacific Biosciences (PacBio)’s isoform sequencing and analyzed the transcriptome structure. In total, 1,536,849 high-quality FL transcripts were obtained through single-molecule real-time (SMRT) sequencing, which were then corrected using Illumina sequencing data. Next, 89,666 nonredundant FL transcripts were generated after mapping to the reference genome of P. sinensis; 291 fusion genes and 17,366 novel isoforms were successfully annotated using data from the nonredundant protein sequence database (NR), eukaryotic orthology groups (KOG), the Gene Ontology (GO) project, and the KEGG Orthology (KO) database. Additionally, 19,324 alternative polyadenylation sites, 101,625 alternative splicing events, 12,392 long noncoding RNAs, and 5916 transcription factors were identified. Smad4, Wif1, and 17-β-hsd were identified as female-biased genes, while Nkd2 and Prp18 held a higher expression level in males than females. In summary, we found differences between male and female P. sinensis individuals in AS, lncRNA, genes, and transcripts, which relate to the Wnt pathway, oocyte meiosis, and the TGF-β pathway. Female-biased genes such as Smad4, Wif1, and 17-β-hsd and male-biased genes such as Nkd2 and Prp18 played important roles in the sex determination of P. sinensis. FL transcripts are a precious resource for characterizing the transcriptome of P. sinensis, laying the foundation for further research on the sex-determination mechanisms of P. sinensis.

Full-length transcriptome analysis revealed that 2,4-dichlorophenoxyacetic acid promoted in vitro bulblet initiation in lily by affecting carbohydrate metabolism and auxin signaling.

Bulblet initiation, including adventitious bud initiation and bulblet formation, is a crucial process for lily and other bulbous flowers that are commercially propagated by vegetative means. Here, by a hybrid strategy combining Pacific Biosciences (PacBio) full-length sequencing and Illumina RNA sequencing (RNA-seq), high-quality transcripts of L. brownii (Lb) and its variety, L. brownii var. giganteum (Lbg), during in vitro bulblet initiation were obtained. A total of 53,576 and 65,050 high-quality non-redundant full-length transcripts of Lbg and Lb were generated, respectively. Morphological observation showed that Lbg possessed a stronger capacity to generate bulblets in vitro than Lb, and 1 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) significantly increased bulblet regeneration rate in two lilies. Screening of differentially expressed transcripts (DETs) between different stages and Mfuzz analysis showed 0 DAT to 1 DAT was the crucial stage with the most complex transcriptional change, with carbohydrate metabolism pathway was significantly enriched. In addition, 6,218 and 8,965 DETs were screened between the 2,4-D-treated group and the control group in Lbg and Lb, respectively. 2,4-D application had evident effects on the expression of genes involved in auxin signaling pathway, such as TIRs, ARFs, Aux/IAAs, GH3s and SAURs. Then, we compared the expression profiles of crucial genes of carbohydrate metabolism between different stages and different treatments. SUSs, SUTs, TPSs, AGPLs, GBSSs and SSs showed significant responses during bulblet initiation. The expression of CWINs, SUTs and SWEETs were significantly upregulated by 2,4-D in two lilies. In addition, 2,4-D increased the expression of starch degradation genes (AMYs and BAMs) and inhibited starch synthesis genes (AGPLs, GBSSs and SSs). SBEs were significantly upregulated in Lbg but not in Lb. Significant co-expression was showed between genes involved in carbohydrate metabolism and auxin signaling, together with transcription factors such as bHLHs, MYBs, ERFs and C3Hs. This study indicates the coordinate regulation of bulblet initiation by carbohydrate metabolism and auxin signaling, serving as a basis for further studies on the molecular mechanism of bulblet initiation in lily and other bulbous flowers.

Iso-seq and RNA-seq profiling of Fenneropenaeus penicillatus: Unravelling the signaling pathway and genes involved in the ovarian development

The impediment of inadequate shrimp gonad development has adversely affected their production in the aquaculture sector. One promising strategy to enhance the quality and timing of offspring for commercial purposes is the regulation of their reproductive activity. Nevertheless, the availability of information pertaining to ovarian development in Fenneropenaeus penicillatus remains limited. To address this knowledge gap, our study employed a multi-faceted approach, integrating cutting-edge technologies such as Iso-seq, RNA-seq, RT-qPCR, histological and morphological observations. The Iso-seq generating 81,964,598 subreads with an average length of 1600 bp. This effort yielded 926,713 CCS reads and 557,043 FLNC reads, culminating in a robust set of 16,587 non-redundant full-length transcripts. This study utilized CPC, CPAT, PLEK, and pfam, to predict the numbers of long non-coding RNAs (lncRNAs). The outcomes were as follows: 5204, 4478, 7055, and 8175 lncRNAs, respectively. Our findings unveiled the intricate molecular underpinnings of ovarian development, encompassing pivotal pathways such as oocyte meiosis, insulin signaling pathway, oxytocin signaling pathway, progesterone-mediated oocyte maturation, estrogen signaling pathway, and GnRH signaling pathway. Notably, we identified specific genes—including Vtg, VgR, Sxl4, UCH, Hsp70, PP1, CDK2, Rheb, MKK3, and CDC42—that exhibited dynamic expression patterns during ovarian development. This study has produced a comprehensive dataset of full-length transcripts that holds significant potential as a genomic reference for future research on F. penicillatus. Furthermore, these insights may substantially contribute to the advancement of artificial breeding techniques and the ongoing development of sustainable aquaculture practices for F. penicillatus.

A comparative full-length transcriptomic resource provides insight into the perennial monocarpic mass flowering.

Perennial monocarpic mass flowering represents as a key developmental innovation in flowering time diversity in several biological and economical essential families, such as the woody bamboos and the shrubby Strobilanthes. However, molecular and genetic mechanisms underlying this important biodiversity remain poorly investigated. Here, we generated a full-length transcriptome resource incorporated into the BlueOmics database (http://blueomics.iflora.cn) for two Strobilanthes species, which feature contrasting flowering time behaviors. Using about 112 and 104 Gb Iso-seq reads together with ~185 and ~75 Gb strand-specific RNA seq data, we annotated 80 971 and 79 985 non-redundant full-length transcripts for the perennial polycarpic Strobilanthes tetrasperma and the perennial monocarpic Strobilanthes biocullata, respectively. In S. tetrasperma, we identified 8794 transcripts showing spatiotemporal expression in nine tissues. In leaves and shoot apical meristems at two developmental stages, 977 and 1121 transcripts were differentially accumulated in S. tetrasperma and S. biocullata, respectively. Interestingly, among the 33 transcription factors showing differential expression in S. tetrasperma but without differential expression in S.biocullata, three were involved potentially in the photoperiod and circadian-clock pathway of flowering time regulation (FAR1 RELATED SEQUENCE 12, FRS12; NUCLEAR FACTOR Y A1, NFYA1; PSEUDO-RESPONSE REGULATOR 5, PRR5), hence provides an important clue in deciphering the flowering diversity mechanisms. Our data serve as a key resource for further dissection of molecular and genetic mechanisms underpinning key biological innovations, here, the perennial monocarpic mass flowering.

Identification and characterization of Crocus sativus WRKY and its interacting MPK involved in crocins biosynthesis based on full-length transcriptome analysis

Crocus sativus, known as “plant gold”, is rich in carotenoids/apocarotenoids and possesses magnificent medicinal properties. Methyl jasmonate (MeJA) can remarkably enhance the production of carotenoids/apocarotenoids in C. sativus, but its regulatory network remains little understood. Herein, MeJA-induced saffron was sequenced using the combination of third generation long-read and next-generation short-read technology. A total of 75,649 high-quality non-redundant full-length transcripts were generated. Functional annotation of differentially expressed genes (DEGs) showed that post-translational modification was crucial to MeJA-induced saffron. In addition, most of the transcripts encoding enzymes in pathways were up-regulated which in accord with the increasing tendency of crocins content. Then 40 CsWRKYs, 19 CsMPKs were identified with in-depth sequence analysis and established the co-expression network of MPK-WRKY-enzyme through mathematical calculation. Finally, RT-qPCR was performed and a total of 2 CsWRKYs (CsWRKY38, 34) and 1 CsMPK (CsMPK6) with significant correlation were identified as good candidates for carotenoids/apocarotenoids biosynthetic regulation. Our results provide inspiration for blank research on post-translational modification of crocins biosynthesis in C. sativus.

Deciphering the regulatory network of miR156 in plant architecture and abiotic stress resistance of alfalfa (Medicago sativa) by transcriptome sequencing

To improve alfalfa (Medicago sativa) biomass yield and abiotic stress resistance is a challenge due to its autotetraploidy and self-incompatibility. Previously we have generated transgenic alfalfa lines overexpressing miR156 and showing excellent plant architecture. In this study, we investigated the regulation network of miR156 in these transgenic alfalfa lines. Based on 83,202 non-redundant full-length transcripts in a pool of roots, stems, leaves, flowers, pods, and apical buds, we identified 154 candidate genes as potential downstream targets of miR156, especially for M. sativa SQUAMOSA promoter binding protein-like genes (MsSPLs), and then identified 26 MsSPL gene family members in alfalfa genome. The expressions of MsSPL5B and MsSPL3 were downregulated in root and leaf respectively, whereas that of MsSPL4 was significantly downregulated in stem, leaf, root, and apical bud tissues due to miR156 overexpression. We revealed the key role of MsSPL4-mediated regulatory pathways in the substantial correlations between agronomic traits and gene expression, and further found several involved hub genes showing significant correlation with MsSPL4. In addition, MsSPL4 overexpression in tobacco enhanced salinity tolerance but inhibited bud induction. Our results provide strong evidence for understanding the roles of miR156 and MsSPLs in regulating plant architecture and abiotic stress responses in alfalfa.

Integrated full-length transcriptome and metabolome analysis of the early defense mechanisms in Paulownia witches’ broom disease

ABSTRACT Paulownia witches’ broom (PaWB) is a devastating disease of Paulownia spp. caused by phytoplasma infection. To reveal the reason for the absence of morphological changes at the early stage of phytoplasma infection, in this study, a combination of second-and third-generation sequencing (single-molecule real-time long-read isoform) and liquid chromatography tandem mass spectrometry were performed to analyse the changes of full-length transcripts and metabolites at the early stage of phytoplasma infection. A total of 140,528 non-redundant full-length transcripts, 597 transcription factors, 1,658 long non-coding RNAs, and 645 metabolites were identified. The third-generation sequencing improved the annotation of the reference Paulownia fortunei (Seem.) Hemsl. genome by 8.86%. Combination analysis of the full-length transcriptome and metabolome revealed that phenolics and phenylalanine ammonia-lyase synthesis related genes were significantly up-regulated in the early stage of phytoplasma-infected P. fortunei. Based on the results of full-length transcriptome and metabolome integrative analysis, we constructed an early defence model diagram of PaWB disease. Our results provide powerful insights into the reason for absence of morphological at the early stage of phytoplasma infection.

Single-molecule Real-time (SMRT) Sequencing Facilitates Transcriptome Research and Genome Annotation of the Fish Sillago sinica.

As a newly described Sillaginidae species, Chinese sillago (Sillago sinica) needs a better understanding of gene annotation information. In this study, we reported the first full-length transcriptome data of S. sinica using the PacBio isoform sequencing Iso-seq and a description of transcriptome structure analysis. A total of 454,979 high-quality full-length transcripts were obtained by single-molecule real-time (SMRT) sequencing, which was corrected by Illumina sequencing data. After that, 66,948 non-redundant full-length transcripts were generated after mapping to the reference genome of S. sinica, including 49 fusion isoforms and 9,250 novel isoforms. 63,459 isoforms were successfully annotated by one of the Nr, Nt, SwissProt, Pfam, KOG, GO, and KEGG databases. Additionally, 30,987 alternative polyadenylation (APA) sites, 451,867 alternative splicing (AS) events, 21,928 long non-coding RNAs (lncRNAs) and 12,911 transcription factors (TFs) were identified. The full-length transcripts of S. sinica would provide a precious resource for characterizing the transcriptome of S. sinica and for the further study of gene function and regulatory mechanism of this species.

Full-length transcriptome-referenced analysis reveals crucial roles of hormone and wounding during induction of aerial bulbils in lily

Aerial bulbils are important vegetative reproductive organs in Lilium. They are often perpetually dormant in most Lilium species, and little is known about the induction of these vegetative structures. The world-famous Oriental hybrid lily cultivar ‘Sorbonne’, which blooms naturally devoid of aerial bulbils, is known for its lovely appearance and sweet fragrance. We found that decapitation stimulated the outgrowth of aerial bulbils at lower stems (LSs) and then application of low and high concentrations of IAA promoted aerial bulbils emergence around the wound at upper stems (USs) of ‘Sorbonne’. However, the genetic basis of aerial bulbil induction is still unclear. Herein, ‘Sorbonne’ transcriptome has been sequenced for the first time using the combination of third-generation long-read and next-generation short-read technology. A total of 46,557 high-quality non-redundant full-length transcripts were generated. Transcriptomic profiling was performed on seven tissues and stems with treatments of decapitation and application of low and high concentrations of IAA, respectively. Functional annotation of 1918 DEGs within stem samples of different treatments showed that hormone signaling, sugar metabolism and wound-induced genes were crucial to bulbils outgrowth. The expression pattern of auxin-, shoot branching hormone-, plant defense hormone- and wound-inducing-related genes indicated their crucial roles in bulbil induction. Then we established five hormone- and wounding-regulated co-expression modules and identified some candidate transcriptional factors, such as MYB, bZIP, and bHLH, that may function in inducing bulbils. High connectivity was observed among hormone signaling genes, wound-induced genes, and some transcriptional factors, suggesting wound- and hormone-invoked signals exhibit extensive cross-talk and regulate bulbil initiation-associated genes via multilayered regulatory cascades. We propose that the induction of aerial bulbils at LSs after decapitation can be explained as the release of apical dominance. In contrast, the induction of aerial bulbils at the cut surface of USs after IAA application occurs via a process similar to callus formation. This study provides abundant candidate genes that will deepen our understanding of the regulation of bulbil outgrowth, paving the way for further molecular breeding of lily.

Integrated Full-Length Transcriptome and MicroRNA Sequencing Approaches Provide Insights Into Salt Tolerance in Mangrove (Sonneratia apetala Buch.-Ham.).

MicroRNAs (miRNAs) are small RNA molecules that serve as key players in plant stress responses. Although stress-regulated miRNAs have been explored in various plants, they are not well studied in mangroves. Herein, we combined PacBio isoform sequencing (Iso-Seq) with BGISEQ short-read RNA-seq to probe the role of miRNAs in the salt stress response of the mangrove plant, Sonneratia apetala Buch.-Ham. A total of 1,702,463 circular consensus sequencing reads were generated that produced 295,501 nonredundant full-length transcripts from the leaves of a 1-year-old S. apetala. After sequencing nine small RNA libraries constructed from control and 1- and 28-day 300 mM NaCl treatments, we identified 143 miRNAs (114 known and 29 novel) from a total of >261 million short reads. With the criteria of |log2FC| ≥ 1 and q-value < 0.05, 42 and 70 miRNAs were differentially accumulated after 1- and 28-day salt treatments, respectively. These differential accumulated miRNAs potentially targeted salt-responsive genes encoding transcription factors, ion homeostasis, osmotic protection, and detoxificant-related proteins, reminiscent of their responsibility for salinity adaptation in S. apetala. Particularly, 62 miRNAs were Sonneratia specific under salt stress, of which 34 were co-expressed with their 131 predicted targets, thus producing 140 miRNA–target interactions. Of these, 82 miRNA-target pairs exhibited negative correlations. Eighteen miRNA targets were categorized for the ‘environmental information processing’ during KEGG analysis and were related to plant hormone signal transduction (ko04075), MAPK signaling pathway–plant (ko04016), and ABC transporters (ko02010). These results underscored miRNAs as possible contributors to mangrove success in severe environments and offer insights into an miRNA-mediated regulatory mechanism of salt response in S. apetala.

Full-Length Transcriptome Analysis Provides New Insights Into the Diversity of Immune-Related Genes in Portunus trituberculatus.

Generally, invertebrates were thought to solely rely on their non-specific innate immune system to fight against invading microorganisms. However, increasing studies have implied that the innate immune response of invertebrates displayed diversity and specificity owing to the hyper-variable immune molecules in organisms. In order to get an insight into the diversity of immune-related genes in Portunus trituberculatus, a full-length transcriptome analysis of several immune-related tissues (hemocytes, hepatopancreas and gills) in P. trituberculatus was performed and the diversity of several immune-related genes was analyzed. The full-length transcriptome analysis of P. trituberculatus was conducted using a combination of SMRT long-read sequencing and Illumina short-read sequencing. A total of 17,433 nonredundant full-length transcripts with average length of 2,271 bp and N50 length of 2,841 bp were obtained, among which 13,978 (80.18%) transcripts were annotated. Moreover, numerous transcript variants of various immune-related genes were identified, including pattern recognition receptors, antimicrobial peptides, heat shock proteins (HSPs), antioxidant enzymes and vital molecules in prophenoloxidase (proPO)-activating system. Based on the full-length transcriptome analysis, open reading frames (ORFs) of four C-type lectins (CTLs) were cloned, and tissue distributions showed that the four CTLs were ubiquitously expressed in all the tested tissues, and mainly expressed in hepatopancreas and gills. The transcription of the four CTLs significantly increased in several immune-related tissues (hemocytes, hepatopancreas and gills) of P. trituberculatus challenged with Vibrio alginolyticus and displayed different profiles. Moreover, the four CTLs displayed distinct bacterial binding and antibacterial activities. The recombinant protein PtCTL-1 (rPtCTL-1) and rPtCTL-3 displayed bacterial binding and antibacterial activities against all tested bacteria. rPtCTL-2 only showed bacterial binding and antibacterial activities against V. alginolyticus. No obvious bacterial binding or antibacterial activities for PtCTL-4 was observed against the tested bacteria. This study enriches the transcriptomic information on P. trituberculatus and provides new insights into the innate immune system of crustaceans. Additionally, our study provided candidates of antibiotic agents for the prevention and treatment of bacteriosis.

Dynamic Responses of Antioxidant and Glyoxalase Systems to Seed Aging Based on Full-Length Transcriptome in Oat (Avena sativa L.).

Seed aging is a major challenge for food security, agronomic production, and germplasm conservation, and reactive oxygen species (ROS) and methylglyoxal (MG) are highly involved in the aging process. However, the regulatory mechanisms controlling the abundance of ROS and MG are not well characterized. To characterize dynamic response of antioxidant and glyoxalase systems during seed aging, oat (Avena sativa L.) aged seeds with a range of germination percentages were used to explore physiological parameters, biochemical parameters and relevant gene expression. A reference transcriptome based on PacBio sequencing generated 67,184 non-redundant full-length transcripts, with 59,050 annotated. Subsequently, eleven seed samples were used to investigate the dynamic response of respiration, ROS and MG accumulation, antioxidant enzymes and glyoxalase activity, and associated genes expression. The 48 indicators with high correlation coefficients were divided into six major response patterns, and were used for placing eleven seed samples into four groups, i.e., non-aged (Group N), higher vigor (Group H), medium vigor (Group M), and lower vigor (Group L). Finally, we proposed a putative model for aging response and self-detoxification mechanisms based on the four groups representing different aging levels. In addition, the outcomes of the study suggested the dysfunction of antioxidant and glyoxalase system, and the accumulation of ROS and MG definitely contribute to oat seed aging.

Dissection of full-length transcriptome and metabolome of Dichocarpum (Ranunculaceae): implications in evolution of specialized metabolism of Ranunculales medicinal plants.

Several main families of Ranunculales are rich in alkaloids and other medicinal compounds; many species of these families are used in traditional and folk medicine. Dichocarpum is a representative medicinal genus of Ranunculaceae, but the genetic basis of its metabolic phenotype has not been investigated, which hinders its sustainable conservation and utilization. We use the third-generation high-throughput sequencing and metabolomic techniques to decipher the full-length transcriptomes and metabolomes of five Dichocarpum species endemic in China, and 71,598 non-redundant full-length transcripts were obtained, many of which are involved in defense, stress response and immunity, especially those participating in the biosynthesis of specialized metabolites such as benzylisoquinoline alkaloids (BIAs). Twenty-seven orthologs extracted from trancriptome datasets were concatenated to reconstruct the phylogenetic tree, which was verified by the clustering analysis based on the metabolomic profile and agreed with the Pearson correlation between gene expression patterns of Dichocarpum species. The phylogenomic analysis of phytometabolite biosynthesis genes, e.g., (S)-norcoclaurine synthase, methyltransferases, cytochrome p450 monooxygenases, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, revealed the evolutionary trajectories leading to the chemodiversity, especially that of protoberberine type, aporphine type and bis-BIA abundant in Dichocarpum and related genera. The biosynthesis pathways of these BIAs are proposed based on full-length transcriptomes and metabolomes of Dichocarpum. Within Ranunculales, the gene duplications are common, and a unique whole genome duplication is possible in Dichocarpum. The extensive correlations between metabolite content and gene expression support the co-evolution of various genes essential for the production of different specialized metabolites. Our study provides insights into the transcriptomic and metabolomic landscapes of Dichocarpum, which will assist further studies on genomics and application of Ranunculales plants.

Global transcriptome landscape of the rabbit protozoan parasite Eimeria stiedae

BackgroundCoccidiosis caused by Eimeria stiedae is a widespread and economically significant disease of rabbits. The lack of studies on the life-cycle development and host interactions of E. stiedae at the molecular level has hampered our understanding of its pathogenesis.MethodsIn this study, we present a comprehensive transcriptome landscape of E. stiedae to illustrate its dynamic development from unsporulated oocysts to sporulated oocysts, merozoites, and gametocytes, and to identify genes related to parasite-host interactions during parasitism using combined PacBio single-molecule real-time and Illumina RNA sequencing followed by bioinformatics analysis and qRT-PCR validation.ResultsIn total, 12,582 non-redundant full-length transcripts were generated with an average length of 1808 bp from the life-cycle stages of E. stiedae. Pairwise comparisons between stages revealed 8775 differentially expressed genes (DEGs) showing highly significant description changes, which compiled a snapshot of the mechanisms underlining asexual and sexual biology of E. stiedae including oocyst sporulation between unsporulated and sporulated oocysts; merozoite replication between sporulated oocysts and merozoites; and gametophyte development and gamete generation between merozoites and gametocytes. Further, 248 DEGs were grouped into nine series clusters and five groups by expression patterns, and showed that parasite–host interaction-related genes predominated in merozoites and gametocytes and were mostly involved in steroid biosynthesis and lipid metabolism and carboxylic acid. Additionally, co-expression analyses identified genes associated with development and host invasion in unsporulated and sporulated oocysts and immune interactions during gametocyte parasitism.ConclusionsThis is the first study, to our knowledge, to use the global transcriptome profiles to decipher molecular changes across the E. stiedae life cycle, and these results not only provide important information for the molecular characterization of E. stiedae, but also offer valuable resources to study other apicomplexan parasites with veterinary and public significance.Graphical

Nanopore-based full-length transcriptome sequencing of Muscovy duck (Cairina moschata) ovary

Unlike mammals, studies on mechanisms that regulate waterfowl ovulation have been rarely reported. To advance our understanding of the ovulation differences in Muscovy duck, we utilized the Oxford Nanopore Technologies (ONT) to generate transcriptome data from 3 groups of female duck ovaries with ovulation differences (i.e., preovulation [PO], consecutive ovulation [CO], and inconsecutive ovulation [IO]). In this study, the full-length transcriptome data qualitative analysis showed that a total of 24,504 nonredundant full-length transcripts were generated, 19,060 new transcripts were discovered and 14,848 novel transcripts were successfully annotated. For the quantitative analysis, differentially expressed genes (DEGs) between the 3 groups were identified and functional properties were characterized. CTNNB1, IGF1, FOXO3, HSPA2, PTEN and SMC4 may be potential hub genes that regulate ovulation. Adhesion-related pathway, mTOR pathway, TGF-β signaling pathway and FoxO signaling pathway have been considered as important pathways that affect follicular development and ovulation. These results provide a more complete data source of full-length transcriptome for the further study of gene expression and genetics in Muscovy duck. The hub genes and potential mechanisms that affect the ovulation of Muscovy duck have been screened out to provide a scientific basis for breeding work to improve the reproduction performance of Muscovy duck.

Long-read transcriptome sequencing reveals abundant promoter diversity in distinct molecular subtypes of gastric cancer

BackgroundDeregulated gene expression is a hallmark of cancer; however, most studies to date have analyzed short-read RNA sequencing data with inherent limitations. Here, we combine PacBio long-read isoform sequencing (Iso-Seq) and Illumina paired-end short-read RNA sequencing to comprehensively survey the transcriptome of gastric cancer (GC), a leading cause of global cancer mortality.ResultsWe performed full-length transcriptome analysis across 10 GC cell lines covering four major GC molecular subtypes (chromosomal unstable, Epstein-Barr positive, genome stable and microsatellite unstable). We identify 60,239 non-redundant full-length transcripts, of which > 66% are novel compared to current transcriptome databases. Novel isoforms are more likely to be cell line and subtype specific, expressed at lower levels with larger number of exons, with longer isoform/coding sequence lengths. Most novel isoforms utilize an alternate first exon, and compared to other alternative splicing categories, are expressed at higher levels and exhibit higher variability. Collectively, we observe alternate promoter usage in 25% of detected genes, with the majority (84.2%) of known/novel promoter pairs exhibiting potential changes in their coding sequences. Mapping these alternate promoters to TCGA GC samples, we identify several cancer-associated isoforms, including novel variants of oncogenes. Tumor-specific transcript isoforms tend to alter protein coding sequences to a larger extent than other isoforms. Analysis of outcome data suggests that novel isoforms may impart additional prognostic information.ConclusionsOur results provide a rich resource of full-length transcriptome data for deeper studies of GC and other gastrointestinal malignancies.

The PacBio Full-Length Transcriptome of the Tea Aphid as a Reference Resource

The tea aphid, Aphis aurantii, has become one of the destructive pests in tea plantations in the tropics and subtropics. Very few functional studies have so far focused on the developmental and reproductive biology at a molecular level, because of the lack of comprehensive genetic information. Full-length transcriptomes represent a very highly efficient approach to obtain reference gene sequences in non-model insects. In the present study, the transcriptome of A. aurantii was comprehensively sequenced using PacBio Iso-Seq technology. A total of 46.8 Gb nucleotides and 15,938 non-redundant full-length transcripts were obtained, 13,498 (84.69%) of which were annotated into seven databases. Of these transcripts, 2,029 alternative splicing events and 15,223 simple sequence repeats were detected. Among these transcripts, 4,571 (28.68%) and 11,367 (71.32%) were long non-coding RNAs (lncRNAs) and protein-coding genes, respectively. Five hundred and ninety transcription factors were detected. The first full-length transcriptome represents a significant increase in the known genetic information of A. aurantii. It will assist the future functional study of genes involved in its development and reproduction.

Comparative transcriptomic analysis provides insights into the coordinated mechanisms of leaves and roots response to cold stress in Common Vetch

Due to the stronger ability to grow in extreme abiotic stress conditions, common vetch (Vicia sativa L.) has been generally cultivated worldwide and commonly utilized in methane biofuel production and health-promoting foods, and considered as an important protein source to feed livestock and human consumption. Nevertheless, the comprehensive molecular mechanisms on how the aboveground and underground tissues of common vetch response to various abiotic stresses, especially in cold stress, remain largely elusive and unknown. In this study, a total of 30,525 non-redundant full-length transcripts were identified in common vetch, with an average length of 2,291.56 bp and N50 of 3,130 bp. Among these transcripts, 7,689 and 3,415 differentially expressed genes (DEGs) were identified in leaves and roots, respectively, and 1,560 DEGs were induced in both tissues. The Gene Ontology (GO) enrichment results displayed that the DEGs were significantly enriched in the photosynthetic related categories in leaves. Correspondingly, the “response to cold” category was significantly enriched in roots, and most of them were upregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that Ca2+, redox, hormonal signalling, circadian clock, and photosynthesis-antenna proteins might play crucial roles through CBF dependent or independent transcriptional pathways to respond cold stress in common vetch. Besides, a total of 36 DEGs involved in ICE-CBF-COR signalling cascade and cold stress were identified. Furthermore, three cold response-related DEGs and three upregulated transcription factors were overexpressed in yeast and indicated their biological functions to confer the tolerance to cold. This work will advance the understanding of common and distinct intrinsic molecular mechanisms beneath the cold response in common vetch leaf and root tissues. The further investigation of the promising potential candidate genes identified in this study will serve as a valuable resource for next step functional genomics research and raise the possibility to improve the cold tolerance through a transgenic approach in common vetch.

Transcriptome sequencing of cochleae from constant-frequency and frequency-modulated echolocating bats

Echolocating bats are fascinating for their ability to ‘see’ the world in the darkness. Ultrahigh frequency hearing is essential for echolocation. In this study we collected cochlear tissues from constant-frequency (CF) bats (two subspecies of Rhinolophus affinis, Rhinolophidae) and frequency-modulated (FM) bats (Myotis ricketti, Vespertilionidae) and applied PacBio single-molecule real-time isoform sequencing (Iso-seq) technology to generate the full-length (FL) transcriptomes for the three taxa. In total of 10103, 9676 and 10504 non-redundant FL transcripts for R. a. hainanus, R. a. himalayanus and Myotis ricketti were obtained respectively. These data present a comprehensive list of transcripts involved in ultrahigh frequency hearing of echolocating bats including 26342 FL transcripts, 24833 of which are annotated by public databases. No further comparative analyses were performed on the current data in this study. This data can be reused to quantify gene or transcript expression, assess the level of alternative splicing, identify novel transcripts and improve genome annotation of bat species.

Full-length transcriptome analysis provides new insights into the innate immune system of Marsupenaeus japonicus

As invertebrates, shrimp are generally thought to solely rely on their innate immune system to combat invading pathogens. Recently, an increasing number of studies have revealed that the innate immune response of invertebrates exhibits diversity and specificity based on their diverse immune molecules. Herein, a full-length transcriptome analysis of several immune-related tissues (hepatopancreas, gill, hemocytes, stomach and intestine) in the kuruma shrimp (Marsupenaeus japonicus) was conducted to identify immune-related molecules with a focus on transcript variations. In total, 11,222 nonredundant full-length transcripts with an N50 length of 5174 were obtained, and most of these transcripts (94.84%) were successfully annotated. In addition, a total of 147 long noncoding RNAs (lncRNAs) were also predicted. Importantly, transcript variants of several vital immune-related genes were observed, including twenty-five alpha-2-macroglobulins (α2-Ms), ten Toll-like receptors (TLRs), six C-type lectins (CTLs), five M-type lectins (MTLs) and three Down syndrome cell adhesion molecules (Dscams). Furthermore, 509 nonredundant full-length transcripts were predicted to be generated from alternative splicing (AS) events, which contribute to the diversity of immune molecules. Overall, our study provides valuable data on the full-length transcripts of M. japonicus, which will facilitate the exploration of immune molecules in this species. Moreover, numerous transcript variants of immune molecules detected in this study provide clues for further investigating the diversity and specificity of the innate immune response in shrimp.