Transcriptome Annotation Research Articles

Comprehensive Characterization of Multitissue Expression Landscape, Co-Expression Networks and Positive Selection in Pikeperch.

Promising efforts are ongoing to extend genomics resources for pikeperch (Sander lucioperca), a species of high interest for the sustainable European aquaculture sector. Although previous work, including reference genome assembly, transcriptome sequence, and single-nucleotide polymorphism genotyping, added a great wealth of genomic tools, a comprehensive characterization of gene expression across major tissues in pikeperch still remains an unmet research need. Here, we used deep RNA-Sequencing of ten vital tissues collected in eight animals to build a high-confident and annotated trancriptome atlas, to detect the tissue-specificity of gene expression and co-expression network modules, and to investigate genome-wide selective signatures in the Percidae fish family. Pathway enrichment and protein–protein interaction network analyses were performed to characterize the unique biological functions of tissue-specific genes and co-expression modules. We detected strong functional correlations and similarities of tissues with respect to their expression patterns—but also significant differences in the complexity and composition of their transcriptomes. Moreover, functional analyses revealed that tissue-specific genes essentially play key roles in the specific physiological functions of the respective tissues. Identified network modules were also functionally coherent with tissues’ main physiological functions. Although tissue specificity was not associated with positive selection, several genes under selection were found to be involved in hypoxia, immunity, and gene regulation processes, that are crucial for fish adaption and welfare. Overall, these new resources and insights will not only enhance the understanding of mechanisms of organ biology in pikeperch, but also complement the amount of genomic resources for this commercial species.

PacBio Single-Molecule Long-Read Sequencing Provides New Light on the Complexity of Full-Length Transcripts in Cattle.

Cattle (Bos taurus) is one of the most widely distributed livestock species in the world, and provides us with high-quality milk and meat which have a huge impact on the quality of human life. Therefore, accurate and complete transcriptome and genome annotation are of great value to the research of cattle breeding. In this study, we used error-corrected PacBio single-molecule real-time (SMRT) data to perform whole-transcriptome profiling in cattle. Then, 22.5 Gb of subreads was generated, including 381,423 circular consensus sequences (CCSs), among which 276,295 full-length non-chimeric (FLNC) sequences were identified. After correction by Illumina short reads, we obtained 22,353 error-corrected isoforms. A total of 305 alternative splicing (AS) events and 3,795 alternative polyadenylation (APA) sites were detected by transcriptome structural analysis. Furthermore, we identified 457 novel genes, 120 putative transcription factors (TFs), and 569 novel long non-coding RNAs (lncRNAs). Taken together, this research improves our understanding and provides new insights into the complexity of full-length transcripts in cattle.

Gene Annotation and Transcriptome Delineation on a De Novo Genome Assembly for the Reference Leishmania major Friedlin Strain.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

Physiological and transcriptome analysis elucidates the metabolic mechanism of versatile Porphyridium purpureum under nitrogen deprivation for exopolysaccharides accumulation

Porphyridium purpureum is a mesophilic, unicellular red alga rich in phycoerythrin, sulfate polysaccharides, and polyunsaturated fatty acids. Nitrogen deficiency inhibited the growth of P. purpureum and resulted in yellowing of the cells and thickening of the extracellular viscousness sheath. Under nitrogen stress, the contents of total lipids and exopolysaccharides in P. purpureum were increased by 65.2% and 188.0%, respectively. We demonstrate that the immediate response of P. purpureum to nitrogen deficiency is mediated by carbon flow to polysaccharide synthesis, while the synthesis of lipids is enhanced as a permanent energy storage substance at the later stage. Based on transcriptome annotation information, we elucidate the synthesis pathway of polysaccharides from P. purpureum from the perspective of glycosyl-donor interconversion, and demonstrate that the n-6 pathway is the main synthesis pathway of polyunsaturated fatty acids. This study not only provides a production strategy for polysaccharides and fatty acids by single-celled marine red algae P. purpureum, but also provides targets for further genetic modification.

Gene selection for studying frugivore-plant interactions: a review and an example using Queensland fruit fly in tomato.

Fruit production is negatively affected by a wide range of frugivorous insects, among them tephritid fruit flies are one of the most important. As a replacement for pesticide-based controls, enhancing natural fruit resistance through biotechnology approaches is a poorly researched but promising alternative. The use of quantitative reverse transcription PCR (RT-qPCR) is an approach to studying gene expression which has been widely used in studying plant resistance to pathogens and non-frugivorous insect herbivores, and offers a starting point for fruit fly studies. In this paper, we develop a gene selection pipe-line for known induced-defense genes in tomato fruit, Solanum lycopersicum, and putative detoxification genes in Queensland fruit fly, Bactrocera tryoni, as a basis for future RT-qPCR research. The pipeline started with a literature review on plant/herbivore and plant/pathogen molecular interactions. With respect to the fly, this was then followed by the identification of gene families known to be associated with insect resistance to toxins, and then individual genes through reference to annotated B. tryoni transcriptomes and gene identity matching with related species. In contrast for tomato, a much better studied species, individual defense genes could be identified directly through literature research. For B. tryoni, gene selection was then further refined through gene expression studies. Ultimately 28 putative detoxification genes from cytochrome P450 (P450), carboxylesterase (CarE), glutathione S-transferases (GST), and ATP binding cassette transporters (ABC) gene families were identified for B. tryoni, and 15 induced defense genes from receptor-like kinase (RLK), D-mannose/L-galactose, mitogen-activated protein kinase (MAPK), lipoxygenase (LOX), gamma-aminobutyric acid (GABA) pathways and polyphenol oxidase (PPO), proteinase inhibitors (PI) and resistance (R) gene families were identified from tomato fruit. The developed gene selection process for B. tryoni can be applied to other herbivorous and frugivorous insect pests so long as the minimum necessary genomic information, an annotated transcriptome, is available.

Transcriptome annotation in the cloud: complexity, best practices and cost

BACKGROUND The NIH Science and Technology Research Infrastructure for Discovery, Experimentation, and Sustainability (STRIDES) initiative provides NIH-funded researchers cost-effective access to commercial cloud providers, such as Amazon Web Services (AWS) and Google Cloud Platform (GCP). These cloud providers represent an alternative for the execution of large computational biology experiments like transcriptome annotation, which is a complex analytical process that requires the interrogation of multiple biological databases with several advanced computational tools. The core components of annotation pipelines published since 2012 are BLAST sequence alignments using annotated databases of both nucleotide or protein sequences almost exclusively with networked on-premises compute systems. FINDINGS We compare multiple BLAST sequence alignments using AWS and GCP. We prepared several Jupyter Notebooks with all the code required to submit computing jobs to the batch system on each cloud provider. We consider the consequence of the number of query transcripts in input files and the effect on cost and processing time. We tested compute instances with 16, 32, and 64 vCPUs on each cloud provider. Four classes of timing results were collected: the total run time, the time for transferring the BLAST databases to the instance local solid-state disk drive, the time to execute the CWL script, and the time for the creation, set-up, and release of an instance. This study aims to establish an estimate of the cost and compute time needed for the execution of multiple BLAST runs in a cloud environment. CONCLUSIONS We demonstrate that public cloud providers are a practical alternative for the execution of advanced computational biology experiments at low cost. Using our cloud recipes, the BLAST alignments required to annotate a transcriptome with ∼500,000 transcripts can be processed in <2 hours with a compute cost of ∼$200-$250. In our opinion, for BLAST-based workflows, the choice of cloud platform is not dependent on the workflow but, rather, on the specific details and requirements of the cloud provider. These choices include the accessibility for institutional use, the technical knowledge required for effective use of the platform services, and the availability of open source frameworks such as APIs to deploy the workflow.

Transcriptome profiling in susceptible and tolerant rubber tree clones in response to cassiicolin Cas1, a necrotrophic effector from Corynespora cassiicola.

Corynespora cassiicola, a fungal plant pathogen with a large host range, causes important damages in rubber tree (Hevea brasiliensis), in Asia and Africa. A small secreted protein named cassiicolin was previously identified as a necrotrophic effector required for the virulence of C. cassiicola in specific rubber tree clones. The objective of this study was to decipher the cassiicolin-mediated molecular mechanisms involved in this compatible interaction. We comparatively analyzed the RNA-Seq transcriptomic profiles of leaves treated or not with the purified cassiicolin Cas1, in two rubber clones: PB260 (susceptible) and RRIM600 (tolerant). The reads were mapped against a synthetic transcriptome composed of all available transcriptomic references from the two clones. Genes differentially expressed in response to cassiicolin Cas1 were identified, in each clone, at two different time-points. After de novo annotation of the synthetic transcriptome, we analyzed GO enrichment of the differentially expressed genes in order to elucidate the main functional pathways impacted by cassiicolin. Cassiicolin induced qualitatively similar transcriptional modifications in both the susceptible and the tolerant clones, with a strong negative impact on photosynthesis, and the activation of defense responses via redox signaling, production of pathogenesis-related protein, or activation of the secondary metabolism. In the tolerant clone, transcriptional reprogramming occurred earlier but remained moderate. By contrast, the susceptible clone displayed a late but huge transcriptional burst, characterized by massive induction of phosphorylation events and all the features of a hypersensitive response. These results confirm that cassiicolin Cas1 is a necrotrophic effector triggering a hypersensitive response in susceptible rubber clones, in agreement with the necrotrophic-effector-triggered susceptibility model.

Annotation of Novel Dynamic UTRs as Functional Targets of miRNAs During Cardiac Development

Small noncoding microRNAs (miRNAs) play essential roles in post-transcriptional gene regulation in development and disease, predominantly through interactions with mRNA untranslated regions (UTRs). However, the dynamic and tissue specific expression of miRNAs and target UTRs throughout development is poorly characterized. In order to understand the RNA regulatory events that drive cardiac morphogenesis, we captured a simultaneous small RNA and total RNA-seq time course in zebrafish hearts. We extracted de novo transcriptome annotation of dynamically changing UTRs, revealing over ten thousand significantly altered transcripts, with novel 5’ and 3’UTRs for approximately half of all protein coding genes, and predicted regulatory miRNA/UTR interactions during development. In addition to miRNA and mRNA transcriptome resources for studies of RNA regulatory events in cardiac development and disease, this study provides a robust bioinformatic pipeline that should facilitate discovery of posttranscriptional regulatory networks in other developmental systems.

Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

A phylogeny-aware approach reveals unexpected venom components in divergent lineages of cone snails.

Marine gastropods of the genus Conus are renowned for their remarkable diversity and deadly venoms. While Conus venoms are increasingly well studied for their biomedical applications, we know surprisingly little about venom composition in other lineages of Conidae. We performed comprehensive venom transcriptomic profiling for Conasprella coriolisi and Pygmaeconus traillii, first time for both respective genera. We complemented reference-based transcriptome annotation by a de novo toxin prediction guided by phylogeny, which involved transcriptomic data on two additional 'divergent' cone snail lineages, Profundiconus, and Californiconus. We identified toxin clusters (SSCs) shared among all or some of the four analysed genera based on the identity of the signal region-a molecular tag present in toxins. In total, 116 and 98 putative toxins represent 29 and 28 toxin gene superfamilies in Conasprella and Pygmaeconus, respectively; about quarter of these only found by semi-manual annotation of the SSCs. Two rare gene superfamilies, originally identified from fish-hunting cone snails, were detected outside Conus rather unexpectedly, so we further investigated their distribution across Conidae radiation. We demonstrate that both these, in fact, are ubiquitous in Conidae, sometimes with extremely high expression. Our findings demonstrate how a phylogeny-aware approach circumvents methodological caveats of similarity-based transcriptome annotation.

Investigation of proline in superficial scald development during low temperature storage of ‘Dangshansuli’ pear fruit

The role of proline in superficial scald development in pear fruit was investigated in this study. During low temperature storage of ‘Dangshansuli’ pear fruit, superficial scald incidence and index accumulated in association with the alternation of α-farnesene and conjugated trienols (CTols). 14 out of 17 free amino acids were identified; proline content gradually decreased, which was consistent with the up-regulation of PbrP5CS activity and down-regulation of PbrProDH activity in its metabolic pathway. A total of 14 genes involved in proline metabolism were identified based on transcriptome annotation with diverse expression profiles. Results from correlation analysis among proline content further indicate that enzyme activity and gene expression profile, PbrProDH2, PbrProDH4, PbrProDH5 &PbrP5CS5 might play a critical role in proline metabolism during low temperature storage of ‘Dangshansuli’ fruit; thus, influencing superficial scald development which was then functionally validated using the transgenic pear fruit. Postharvest 1-MCP and diphenylamine (DPA) treatment inhibited the accumulation of CTols, and thus, mitigated superficial scald development and maintained higher proline content, in association with the up-regulated PbrP5CS activity, PbrP5CS4 &PbrP5CS5 mRNAs as well as the down-regulated PbrProDH activity &PbrProDH4 transcripts. In a further study, we found that exogenous application of proline alleviated superficial scald incidence and index. To that end, the results of our study confirmed that proline was involved in superficial scald development during pear storage.

Abstract 2879: MACC1 overexpression in colorectal cancer is driven by chromosomal instability and is associated with molecular subtype

Abstract Metastasis-Associated in Colon Cancer 1 (MACC1) is a strong prognostic marker for human colorectal cancers (CRC). The gene is a key regulator of the HGF/c-Met pathway and its overexpression is associated with cancer cell proliferation, migration, invasiveness and metastasis. The contexture of MACC1 overexpression in cancers is still poorly understood. Here we used the data released by The Cancer Genome Atlas (TCGA) and in silico approaches for a deep and integrative analysis of MACC1 expression modalities in CRCs. First, we curated COAD-READ TCGA datasets and obtained full clinical, genomic, and transcriptomic annotation for a total of 259 CRC samples. In tumors, we showed high heterogeneity of MACC1 mRNA expression levels with data varying from 5.58 to 13.07 U (RNAseq log transformed data - RSEM normalized). By correlating MACC1 expression to clinical data, we showed its levels were associated with anatomic regions of the disease (p=6.35.10-5). The tumors from descending colon and rectum having higher levels than those of the ascending colon and caecum (p=7.44-06). High MACC1 was also associated with higher tumor stages (p=0.01) and nodal invasion (p=0.03). At genomic level, MACC1 mutation was not frequent in CRCs (&amp;lt;2%). The gene is however frequently subjected to gene copy number variation (CNV) due to chromosomal instability and polyploidy (MACC1 CNV values &amp;gt; normal in 155/259: 60%). Correlation analysis indicated strong positive association between MACC1 copy number variations and mRNA expression levels (p&amp;lt;10-16). In an independent dataset, we validate that gain of MACC1 gene copy number and associated higher expression levels correlated with metastasis dissemination and worse outcome of the patients. By analyzing MACC1 with respect of CRC subtypes, we confirmed higher MACC1 levels in MSS tumors with high chromosomal instability (CIN) compared to MSI or genomic stable tumors (GS) (p=1.16-14). Regarding association with CMS1-4 transcriptomic subtypes, the highest expression of MACC1 were observed in CMS2-(epithelial), and CMS4 (mesenchymal) as compared with CMS3 (epithelial with metabolic dysregulation) or CMS1 (MMR deficient). The subset of tumors with very low MACC1 expression were mainly those altered for MLH1 (MSI-H- MMR tumors), notably most of them have normal ploidy. Deconvolution analysis of immune contexture revealed negative correlation between MACC1 expression and immune activation. Stratification using MACC1 as prognostic marker within these tumor types will be evaluated and the gene will also be presented with respect of alterations of key cancer genes in these tumors. Overall, the present study gives insight into the contexture of MACC1 expression in CRC. We showed MACC1 overexpression is largely driven by chromosomal instability and DNA copy number gains. Its expression was dependent on the tumor molecular subtypes. Citation Format: Vincent Vuaroqueaux, Hoor Al-Hasani, Dennis Kobelt, Thomas Risch, Susen Burock, Marie-Laure Yaspo, Heinz-Herbert Fiebig, Ulrike Stein. MACC1 overexpression in colorectal cancer is driven by chromosomal instability and is associated with molecular subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2879.

Characterization and RNA-seq transcriptomic analysis of a Scenedesmus obliqnus mutant with enhanced photosynthesis efficiency and lipid productivity

Microalgae have received significant attention as potential next-generation microbiologic cell factories for biofuels. However, the production of microalgal biofuels is not yet sufficiently cost-effective for commercial applications. To screen higher lipid-producing strains, heavy carbon ion beams are applied to induce a genetic mutant. An RNA-seq technology is used to identify the pathways and genes of importance related to photosynthesis and biofuel production. The deep elucidation of photosynthesis and the fatty acid metabolism pathway involved in lipid yield is valuable information for further optimization studies. This study provided the photosynthetic efficiency and transcriptome profiling of a unicellular microalgae, Scenedesmus obliqnus mutant SO120G, with enhanced lipid production induced by heavy carbon ion beams. The lipid yield (52.5 mg L−1) of SO120G mutant were enhanced 2.4 fold compared with that of the wild strain under the nitrogen deficient condition. In addition, the biomass and growth rate were 57% and 25% higher, respectively, in SO120G than in the wild type, likely owing to an improved maximum quantum efficiency (Fv/Fm) of photosynthesis. As for the major pigment compositions, the content of chlorophyll a and carotenoids was higher in SO120G than in the wild type. The transcriptome data confirmed that a total of 2077 genes with a change of at least twofold were recognized as differential expression genes (DEGs), of which 1060 genes were up-regulated and 1017 genes were down-regulated. Most of the DEGs involved in lipid biosynthesis were up-regulated with the mutant SO120G. The expression of the gene involved in the fatty acid biosynthesis and photosynthesis of SO120G was upregulated, while that related to starch metabolism decreased compared with that of the wild strain. This work demonstrated that heavy-ion irradiation is an promising strategy for quality improvement. In addition, the mutant SO120G was shown to be a potential algal strain for enhanced lipid production. Transcriptome sequencing and annotation of the mutant suggested the possible genes responsible for lipid biosynthesis and photosynthesis, and identified the putative target genes for future genetic manipulation and biotechnological applications.

TrancriptomeReconstructoR: data-driven annotation of complex transcriptomes

BackgroundThe quality of gene annotation determines the interpretation of results obtained in transcriptomic studies. The growing number of genome sequence information calls for experimental and computational pipelines for de novo transcriptome annotation. Ideally, gene and transcript models should be called from a limited set of key experimental data.ResultsWe developed TranscriptomeReconstructoR, an R package which implements a pipeline for automated transcriptome annotation. It relies on integrating features from independent and complementary datasets: (i) full-length RNA-seq for detection of splicing patterns and (ii) high-throughput 5′ and 3′ tag sequencing data for accurate definition of gene borders. The pipeline can also take a nascent RNA-seq dataset to supplement the called gene model with transient transcripts.We reconstructed de novo the transcriptional landscape of wild type Arabidopsis thaliana seedlings and Saccharomyces cerevisiae cells as a proof-of-principle. A comparison to the existing transcriptome annotations revealed that our gene model is more accurate and comprehensive than the most commonly used community gene models, TAIR10 and Araport11 for A.thaliana and SacCer3 for S.cerevisiae. In particular, we identify multiple transient transcripts missing from the existing annotations. Our new annotations promise to improve the quality of A.thaliana and S.cerevisiae genome research.ConclusionsOur proof-of-concept data suggest a cost-efficient strategy for rapid and accurate annotation of complex eukaryotic transcriptomes. We combine the choice of library preparation methods and sequencing platforms with the dedicated computational pipeline implemented in the TranscriptomeReconstructoR package. The pipeline only requires prior knowledge on the reference genomic DNA sequence, but not the transcriptome. The package seamlessly integrates with Bioconductor packages for downstream analysis.

Large-Scale Multiplexing Permits Full-Length Transcriptome Annotation of 32 Bovine Tissues From a Single Nanopore Flow Cell.

A comprehensive annotation of transcript isoforms in domesticated species is lacking. Especially considering that transcriptome complexity and splicing patterns are not well-conserved between species, this presents a substantial obstacle to genomic selection programs that seek to improve production, disease resistance, and reproduction. Recent advances in long-read sequencing technology have made it possible to directly extrapolate the structure of full-length transcripts without the need for transcript reconstruction. In this study, we demonstrate the power of long-read sequencing for transcriptome annotation by coupling Oxford Nanopore Technology (ONT) with large-scale multiplexing of 93 samples, comprising 32 tissues collected from adult male and female Hereford cattle. More than 30 million uniquely mapping full-length reads were obtained from a single ONT flow cell, and used to identify and characterize the expression dynamics of 99,044 transcript isoforms at 31,824 loci. Of these predicted transcripts, 21% exactly matched a reference transcript, and 61% were novel isoforms of reference genes, substantially increasing the ratio of transcript variants per gene, and suggesting that the complexity of the bovine transcriptome is comparable to that in humans. Over 7,000 transcript isoforms were extremely tissue-specific, and 61% of these were attributed to testis, which exhibited the most complex transcriptome of all interrogated tissues. Despite profiling over 30 tissues, transcription was only detected at about 60% of reference loci. Consequently, additional studies will be necessary to continue characterizing the bovine transcriptome in additional cell types, developmental stages, and physiological conditions. However, by here demonstrating the power of ONT sequencing coupled with large-scale multiplexing, the task of exhaustively annotating the bovine transcriptome – or any mammalian transcriptome – appears significantly more feasible.

Genome sequence, transcriptome, and annotation of rodent malaria parasite Plasmodium yoelii nigeriensis N67

BackgroundRodent malaria parasites are important models for studying host-malaria parasite interactions such as host immune response, mechanisms of parasite evasion of host killing, and vaccine development. One of the rodent malaria parasites is Plasmodium yoelii, and multiple P. yoelii strains or subspecies that cause different disease phenotypes have been widely employed in various studies. The genomes and transcriptomes of several P. yoelii strains have been analyzed and annotated, including the lethal strains of P. y. yoelii YM (or 17XL) and non-lethal strains of P. y. yoelii 17XNL/17X. Genomic DNA sequences and cDNA reads from another subspecies P. y. nigeriensis N67 have been reported for studies of genetic polymorphisms and parasite response to drugs, but its genome has not been assembled and annotated.ResultsWe performed genome sequencing of the N67 parasite using the PacBio long-read sequencing technology, de novo assembled its genome and transcriptome, and predicted 5383 genes with high overall annotation quality. Comparison of the annotated genome of the N67 parasite with those of YM and 17X parasites revealed a set of genes with N67-specific orthology, expansion of gene families, particularly the homologs of the Plasmodium chabaudi erythrocyte membrane antigen, large numbers of SNPs and indels, and proteins predicted to interact with host immune responses based on their functional domains.ConclusionsThe genomes of N67 and 17X parasites are highly diverse, having approximately one polymorphic site per 50 base pairs of DNA. The annotated N67 genome and transcriptome provide searchable databases for fast retrieval of genes and proteins, which will greatly facilitate our efforts in studying the parasite biology and gene function and in developing effective control measures against malaria.

ToxCodAn: a new toxin annotator and guide to venom gland transcriptomics.

Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide. Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time ($>20x$ faster), coding sequence prediction ($>3x$ more accurate) and the number of toxins predicted (generating $>4x$ less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins.

Transcriptomic Analysis of Seasonal Gene Expression and Regulation during Xylem Development in “Shanxin” Hybrid Poplar (Populus davidiana × Populus bolleana)

Xylem development is a key process for wood formation in woody plants. To study the molecular regulatory mechanisms related to xylem development in hybrid poplar P. davidiana × P. bolleana, transcriptome analyses were conducted on developing xylem at six different growth stages within a single growing season. Xylem development and differentially expressed genes in the six time points were selected for a regulatory analysis. Xylem development was observed in stem sections at different growth stages, which showed that xylem development extended from the middle of April to early August and included cell expansion and secondary cell wall biosynthesis. An RNA-seq analysis of six samples with three replicates was performed. After transcriptome assembly and annotation, the differentially expressed genes (DEGs) were identified, and a Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and expression analysis of the DEGs were performed on each sample. On average, we obtained &gt;20 million clean reads per sample, which were assembled into 84,733 nonredundant transcripts, of which there were 17,603 unigenes with lengths &gt;1 kb. There were 14,890 genes that were differentially expressed among the six stages. The upregulated DEGs were enriched in GO terms related to cell wall biosynthesis between S1 vs. S2 or S3 vs. S4 and, in GO terms, related to phytohormones in the S1 vs. S2 or S4 vs. S5 comparisons. The downregulated DEGs were enriched in GO terms related to cell wall biosynthesis between S4 vs. S5 or S5 vs. S6 and, in GO terms, related to hormones between S1 vs. S2 or S2 vs. S3. The KEGG pathways in the DEGs related to “phenylpropanoid biosynthesis”, “plant hormone signal transduction” and “starch and sucrose metabolism” were significantly enriched among the different stages. The DEGs related to cell expansion, polysaccharide metabolism and synthesis, lignin synthesis, transcription factors and hormones were identified. The identification of genes involved in the regulation of xylem development will increase our understanding of the molecular regulation of wood formation in trees and, also, offers potential targets for genetic manipulation to improve the properties of wood.

Functional Annotation of a Full-Length Transcriptome and Identification of Genes Associated with Flower Development in Rhododendronsimsii (Ericaceae).

Rhododendronsimsii is one of the top ten famous flowers in China. Due to its historical value and high aesthetic, it is widely popular among Chinese people. Various colors are important breeding objectives in Rhododendron L. The understanding of the molecular mechanism of flower color formation can provide a theoretical basis for the improvement of flower color in Rhododendron L. To generate the R. simsii transcriptome, PacBio sequencing technology has been used. A total of 833,137 full-length non-chimeric reads were obtained and 726,846 high-quality full-length transcripts were found. Moreover, 40,556 total open reading frames were obtained; of which 36,018 were complete. In gene annotation analyses, 39,411, 18,565, 16,102 and 17,450 transcriptions were allocated to GO, Nr, KEGG and COG databases, correspondingly. 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 6170, 2265, 4084 and 1240 lncRNAs, respectively. Based on the results, most genes were enriched in the flavonoid biosynthetic pathway. The eight key genes on the anthocyanin biosynthetic pathway were further selected and analyzed by qRT-PCR. The F3′H and ANS showed an upward trend in the developmental stages of R. simsii. The highest expression of F3′5′H and FLS in the petal color formation of R. simsii was observed. This research provided a huge number of full-length transcripts, which will help to proceed genetic analyses of R. simsii. native, which is a semi-deciduous shrub.

Gene and Transposable Element Expression Evolution Following Recent and Past Polyploidy Events in Spartina (Poaceae).

Gene expression dynamics is a key component of polyploid evolution, varying in nature, intensity, and temporal scales, most particularly in allopolyploids, where two or more sub-genomes from differentiated parental species and different repeat contents are merged. Here, we investigated transcriptome evolution at different evolutionary time scales among tetraploid, hexaploid, and neododecaploid Spartina species (Poaceae, Chloridoideae) that successively diverged in the last 6–10 my, at the origin of differential phenotypic and ecological traits. Of particular interest are the recent (19th century) hybridizations between the two hexaploids Spartina alterniflora (2n = 6x = 62) and S. maritima (2n = 6x = 60) that resulted in two sterile F1 hybrids: Spartina × townsendii (2n = 6x = 62) in England and Spartina × neyrautii (2n = 6x = 62) in France. Whole genome duplication of S. × townsendii gave rise to the invasive neo-allododecaploid species Spartina anglica (2n = 12x = 124). New transcriptome assemblies and annotations for tetraploids and the enrichment of previously published reference transcriptomes for hexaploids and the allododecaploid allowed identifying 42,423 clusters of orthologs and distinguishing 21 transcribed transposable element (TE) lineages across the seven investigated Spartina species. In 4x and 6x mesopolyploids, gene and TE expression changes were consistent with phylogenetic relationships and divergence, revealing weak expression differences in the tetraploid sister species Spartina bakeri and Spartina versicolor (<2 my divergence time) compared to marked transcriptome divergence between the hexaploids S. alterniflora and S. maritima that diverged 2–4 mya. Differentially expressed genes were involved in glycolysis, post-transcriptional protein modifications, epidermis development, biosynthesis of carotenoids. Most detected TE lineages (except SINE elements) were found more expressed in hexaploids than in tetraploids, in line with their abundance in the corresponding genomes. Comparatively, an astonishing (52%) expression repatterning and deviation from parental additivity were observed following recent reticulate evolution (involving the F1 hybrids and the neo-allododecaploid S. anglica), with various patterns of biased homoeologous gene expression, including genes involved in epigenetic regulation. Downregulation of TEs was observed in both hybrids and accentuated in the neo-allopolyploid. Our results reinforce the view that allopolyploidy represents springboards to new regulatory patterns, offering to worldwide invasive species, such as S. anglica, the opportunity to colonize stressful and fluctuating environments on saltmarshes.