Menu

Asset Tag: RNA sequencing

September 22, 2019

Characterization of novel transcripts in pseudorabies virus.

In this study we identified two 3′-coterminal RNA molecules in the pseudorabies virus. The highly abundant short transcript (CTO-S) proved to be encoded between the ul21 and ul22 genes in close vicinity of the replication origin (OriL) of the virus. The less abundant long RNA molecule (CTO-L) is a transcriptional readthrough product of the ul21 gene and overlaps OriL. These polyadenylated RNAs were characterized by ascertaining their nucleotide sequences with the Illumina HiScanSQ and Pacific Biosciences Real-Time (PacBio RSII) sequencing platforms and by analyzing their transcription kinetics through use of multi-time-point Real-Time RT-PCR and the PacBio RSII system. It emerged that transcription of the CTOs is fully dependent on the viral transactivator protein IE180 and CTO-S is not a microRNA precursor. We propose an interaction between the transcription and replication machineries at this genomic location, which might play an important role in the regulation of DNA synthesis.

Read more
September 22, 2019

Single-Molecule Long-Read Sequencing of Zanthoxylum bungeanum Maxim. Transcriptome: Identification of Aroma-Related Genes

Zanthoxylum bungeanum Maxim. is an economically important tree species that is resistant to drought and infertility, and has potential medicinal and edible value. However, comprehensive genomic data are not yet available for this species, limiting its potential utility for medicinal use, breeding programs, and cultivation. Transcriptome sequencing provides an effective approach to remedying this shortcoming. Herein, single-molecule long-read sequencing and next-generation sequencingapproacheswereusedinparalleltoobtaintranscriptisoformstructureandgenefunctional informationinZ.bungeanum. Intotal, 282,101readsofinserts(ROIs)wereidentified, including134,074 full-length non-chimeric reads, among which 65,711 open reading frames (ORFs), 50,135 simple sequence repeats (SSRs), and 1492 long non-coding RNAs (lncRNAs) were detected. Functional annotation revealed metabolic pathways related to aroma components and color characteristics in Z. bungeanum. Unexpectedly, 30 transcripts were annotated as genes involved in regulating the pathogenesis of breast and colorectal cancers. This work provides a comprehensive transcriptome resource for Z. bungeanum, and lays a foundation for the further investigation and utilization of Zanthoxylum resources.

Read more
September 22, 2019

Extensive alternative splicing of KIR transcripts.

The killer-cell Ig-like receptors (KIR) form a multigene entity involved in modulating immune responses through interactions with MHC class I molecules. The complexity of the KIR cluster is reflected by, for instance, abundant levels of allelic polymorphism, gene copy number variation, and stochastic expression profiles. The current transcriptome study involving human and macaque families demonstrates that KIR family members are also subjected to differential levels of alternative splicing, and this seems to be gene dependent. Alternative splicing may result in the partial or complete skipping of exons, or the partial inclusion of introns, as documented at the transcription level. This post-transcriptional process can generate multiple isoforms from a single KIR gene, which diversifies the characteristics of the encoded proteins. For example, alternative splicing could modify ligand interactions, cellular localization, signaling properties, and the number of extracellular domains of the receptor. In humans, we observed abundant splicing for KIR2DL4, and to a lesser extent in the lineage III KIR genes. All experimentally documented splice events are substantiated by in silico splicing strength predictions. To a similar extent, alternative splicing is observed in rhesus macaques, a species that shares a close evolutionary relationship with humans. Splicing profiles of Mamu-KIR1D and Mamu-KIR2DL04 displayed a great diversity, whereas Mamu-KIR3DL20 (lineage V) is consistently spliced to generate a homolog of human KIR2DL5 (lineage I). The latter case represents an example of convergent evolution. Although just a single KIR splice event is shared between humans and macaques, the splicing mechanisms are similar, and the predicted consequences are comparable. In conclusion, alternative splicing adds an additional layer of complexity to the KIR gene system in primates, and results in a wide structural and functional variety of KIR receptors and its isoforms, which may play a role in health and disease.

Read more
September 22, 2019

Full-length transcriptome survey and expression analysis of Cassia obtusifolia to discover putative genes related to aurantio-obtusin biosynthesis, seed formation and development, and stress response.

The seed is the pharmaceutical and breeding organ of Cassia obtusifolia, a well-known medical herb containing aurantio-obtusin (a kind of anthraquinone), food, and landscape. In order to understand the molecular mechanism of the biosynthesis of aurantio-obtusin, seed formation and development, and stress response of C. obtusifolia, it is necessary to understand the genomics information. Although previous seed transcriptome of C. obtusifolia has been carried out by short-read next-generation sequencing (NGS) technology, the vast majority of the resulting unigenes did not represent full-length cDNA sequences and supply enough gene expression profile information of the various organs or tissues. In this study, fifteen cDNA libraries, which were constructed from the seed, root, stem, leaf, and flower (three repetitions with each organ) of C. obtusifolia, were sequenced using hybrid approach combining single-molecule real-time (SMRT) and NGS platform. More than 4,315,774 long reads with 9.66 Gb sequencing data and 361,427,021 short reads with 108.13 Gb sequencing data were generated by SMRT and NGS platform, respectively. 67,222 consensus isoforms were clustered from the reads and 81.73% (61,016) of which were longer than 1000 bp. Furthermore, the 67,222 consensus isoforms represented 58,106 nonredundant transcripts, 98.25% (57,092) of which were annotated and 25,573 of which were assigned to specific metabolic pathways by KEGG. CoDXS and CoDXR genes were directly used for functional characterization to validate the accuracy of sequences obtained from transcriptome. A total of 658 seed-specific transcripts indicated their special roles in physiological processes in seed. Analysis of transcripts which were involved in the early stage of anthraquinone biosynthesis suggested that the aurantio-obtusin in C. obtusifolia was mainly generated from isochorismate and Mevalonate/methylerythritol phosphate (MVA/MEP) pathway, and three reactions catalyzed by Menaquinone-specific isochorismate synthase (ICS), 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate (IPPS) might be the limited steps. Several seed-specific CYPs, SAM-dependent methyltransferase, and UDP-glycosyltransferase (UDPG) supplied promising candidate genes in the late stage of anthraquinone biosynthesis. In addition, four seed-specific transcriptional factors including three MYB Transcription Factor (MYB) and one MADS-box Transcription Factor (MADS) transcriptional factors) and alternative splicing might be involved with seed formation and development. Meanwhile, most members of Hsp20 genes showed high expression level in seed and flower; seven of which might have chaperon activities under various abiotic stresses. Finally, the expressional patterns of genes with particular interests showed similar trends in both transcriptome assay and qRT-PCR. In conclusion, this is the first full-length transcriptome sequencing reported in Caesalpiniaceae family, and thus providing a more complete insight into aurantio-obtusin biosynthesis, seed formation and development, and stress response as well in C. obtusifolia.

Read more
September 22, 2019

CRISPR/Cas9 deletions in a conserved exon of Distal-less generates gains and losses in a recently acquired morphological novelty in flies.

Distal-less has been repeatedly co-opted for the development of many novel traits. Here, we document its curious role in the development of a novel abdominal appendage (“sternite brushes”) in sepsid flies. CRISPR/Cas9 deletions in the homeodomain result in losses of sternite brushes, demonstrating that Distal-less is necessary for their development. However, deletions in the upstream coding exon (Exon 2) produce losses or gains of brushes. A dissection of Exon 2 reveals that the likely mechanism for gains involves a deletion in an exon-splicing enhancer site that leads to exon skipping. Such contradictory phenotypes are also observed in butterflies, suggesting that mutations in the conserved upstream regions have the potential to generate phenotypic variability in insects that diverged 300 million years ago. Our results demonstrate the importance of Distal-less for the development of a novel abdominal appendage in insects and highlight how site-specific mutations in the same exon can produce contradictory phenotypes. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Read more
September 22, 2019

Lentinula edodes genome survey and postharvest transcriptome analysis.

Lentinula edodes is a popular, cultivated edible and medicinal mushroom. Lentinula edodes is susceptible to postharvest problems, such as gill browning, fruiting body softening, and lentinan degradation. We constructed a de novo assembly draft genome sequence and performed gene prediction for Lentinula edodesDe novo assembly was carried out using short reads from paired-end and mate-paired libraries and by using long reads by PacBio, resulting in a contig number of 1,951 and an N50 of 1 Mb. Furthermore, we predicted genes by Augustus using transcriptome sequencing (RNA-seq) data from the whole life cycle of Lentinula edodes, resulting in 12,959 predicted genes. This analysis revealed that Lentinula edodes lacks lignin peroxidase. To reveal genes involved in the loss of quality of Lentinula edodes postharvest fruiting bodies, transcriptome analysis was carried out using serial analysis of gene expression (SuperSAGE). This analysis revealed that many cell wall-related enzymes are upregulated after harvest, such as ß-1,3-1,6-glucan-degrading enzymes in glycoside hydrolase (GH) families GH5, GH16, GH30, GH55, and GH128, and thaumatin-like proteins. In addition, we found that several chitin-related genes are upregulated, such as putative chitinases in GH family 18, exochitinases in GH20, and a putative chitosanase in GH family 75. The results suggest that cell wall-degrading enzymes synergistically cooperate for rapid fruiting body autolysis. Many putative transcription factor genes were upregulated postharvest, such as genes containing high-mobility-group (HMG) domains and zinc finger domains. Several cell death-related proteins were also upregulated postharvest.IMPORTANCE Our data collectively suggest that there is a rapid fruiting body autolysis system in Lentinula edodes The genes for the loss of postharvest quality newly found in this research will be targets for the future breeding of strains that keep fresh longer than present strains. De novoLentinula edodes genome assembly data will be used for the construction of a complete Lentinula edodes chromosome map for future breeding. Copyright © 2017 American Society for Microbiology.

Read more
September 22, 2019

The habu genome reveals accelerated evolution of venom protein genes.

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

Read more
September 22, 2019

Genomic imprinting mediates dosage compensation in a young plant XY system.

Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced expression and eventual loss of Y genes1. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2-4. However, the early steps in the evolution of dosage compensation remain unknown, and dosage compensation is poorly understood in plants5. Here, we describe a dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males, but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno6 for the evolution of X inactivation in mammals.

Read more
September 22, 2019

PLEK: a tool for predicting long non-coding RNAs and messenger RNAs based on an improved k-mer scheme.

High-throughput transcriptome sequencing (RNA-seq) technology promises to discover novel protein-coding and non-coding transcripts, particularly the identification of long non-coding RNAs (lncRNAs) from de novo sequencing data. This requires tools that are not restricted by prior gene annotations, genomic sequences and high-quality sequencing.We present an alignment-free tool called PLEK (predictor of long non-coding RNAs and messenger RNAs based on an improved k-mer scheme), which uses a computational pipeline based on an improved k-mer scheme and a support vector machine (SVM) algorithm to distinguish lncRNAs from messenger RNAs (mRNAs), in the absence of genomic sequences or annotations. The performance of PLEK was evaluated on well-annotated mRNA and lncRNA transcripts. 10-fold cross-validation tests on human RefSeq mRNAs and GENCODE lncRNAs indicated that our tool could achieve accuracy of up to 95.6%. We demonstrated the utility of PLEK on transcripts from other vertebrates using the model built from human datasets. PLEK attained >90% accuracy on most of these datasets. PLEK also performed well using a simulated dataset and two real de novo assembled transcriptome datasets (sequenced by PacBio and 454 platforms) with relatively high indel sequencing errors. In addition, PLEK is approximately eightfold faster than a newly developed alignment-free tool, named Coding-Non-Coding Index (CNCI), and 244 times faster than the most popular alignment-based tool, Coding Potential Calculator (CPC), in a single-threading running manner.PLEK is an efficient alignment-free computational tool to distinguish lncRNAs from mRNAs in RNA-seq transcriptomes of species lacking reference genomes. PLEK is especially suitable for PacBio or 454 sequencing data and large-scale transcriptome data. Its open-source software can be freely downloaded from https://sourceforge.net/projects/plek/files/.

Read more
September 22, 2019

Lipoprotein lipase reaches the capillary lumen in chickens despite an apparent absence of GPIHBP1.

In mammals, GPIHBP1 is absolutely essential for transporting lipoprotein lipase (LPL) to the lumen of capillaries, where it hydrolyzes the triglycerides in triglyceride-rich lipoproteins. In all lower vertebrate species (e.g., birds, amphibians, reptiles, fish), a gene for LPL can be found easily, but a gene for GPIHBP1 has never been found. The obvious question is whether the LPL in lower vertebrates is able to reach the capillary lumen. Using purified antibodies against chicken LPL, we showed that LPL is present on capillary endothelial cells of chicken heart and adipose tissue, colocalizing with von Willebrand factor. When the antibodies against chicken LPL were injected intravenously into chickens, they bound to LPL on the luminal surface of capillaries in heart and adipose tissue. LPL was released rapidly from chicken hearts with an infusion of heparin, consistent with LPL being located inside blood vessels. Remarkably, chicken LPL bound in a specific fashion to mammalian GPIHBP1. However, we could not identify a gene for GPIHBP1 in the chicken genome, nor could we identify a transcript for GPIHBP1 in a large chicken RNA-seq data set. We conclude that LPL reaches the capillary lumen in chickens – as it does in mammals – despite an apparent absence of GPIHBP1.

Read more
September 22, 2019

Isoform sequencing and state-of-art applications for unravelling complexity of plant transcriptomes

Single-molecule real-time (SMRT) sequencing developed by PacBio, also called third-generation sequencing (TGS), offers longer reads than the second-generation sequencing (SGS). Given its ability to obtain full-length transcripts without assembly, isoform sequencing (Iso-Seq) of transcriptomes by PacBio is advantageous for genome annotation, identification of novel genes and isoforms, as well as the discovery of long non-coding RNA (lncRNA). In addition, Iso-Seq gives access to the direct detection of alternative splicing, alternative polyadenylation (APA), gene fusion, and DNA modifications. Such applications of Iso-Seq facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In this review, we summarize its applications in plant transcriptome study, specifically pointing out challenges associated with each step in the experimental design and highlight the development of bioinformatic pipelines. We aim to provide the community with an integrative overview and a comprehensive guidance to Iso-Seq, and thus to promote its applications in plant research.

Read more
September 22, 2019

Single Molecule Sequencing: new outlooks for solving genome assembly and transcripts identification challenges

In this review, we introduce a novel sequencing technology, named Single Molecule Real Time sequencing. Also called Single Molecule Sequencing, as it do not requires any amplification, this new technology is able to pro- duce much longer reads than previous NGS technologies such as Illumina. This read size improvements, which can reach 150 fold, will solve many challenges caused by the actual NGS technologies. Short NGS reads, reach- ing a maximum size of 300 bp, make it hard to reconstitute a whole genome and are always leading to fragmented genome assembly. It is also difficult to correctly infer transcript quantification and identification when there is a high isoforms diversity. Despite their higher error rate, long reads have shown very promising result concerning these actual issues. We show that longer reads can produce less fragmented assembly, with a better quality, but also sequence from start to end mRNA, making it much more easier to infer correct transcript quantification, and even allow new intron structure and so new isoforms discovery.

Read more
September 22, 2019

Global identification of alternative splicing via comparative analysis of SMRT- and Illumina-based RNA-seq in strawberry.

Alternative splicing (AS) is a key post-transcriptional regulatory mechanism, yet little information is known about its roles in fruit crops. Here, AS was globally analyzed in the wild strawberry Fragaria vesca genome with RNA-seq data derived from different stages of fruit development. The AS landscape was characterized and compared between the single-molecule, real-time (SMRT) and Illumina RNA-seq platform. While SMRT has a lower sequencing depth, it identifies more genes undergoing AS (57.67% of detected multiexon genes) when it is compared with Illumina (33.48%), illustrating the efficacy of SMRT in AS identification. We investigated different modes of AS in the context of fruit development; the percentage of intron retention (IR) is markedly reduced whereas that of alternative acceptor sites (AA) is significantly increased post-fertilization when compared with pre-fertilization. When all the identified transcripts were combined, a total of 66.43% detected multiexon genes in strawberry undergo AS, some of which lead to a gain or loss of conserved domains in the gene products. The work demonstrates that SMRT sequencing is highly powerful in AS discovery and provides a rich data resource for later functional studies of different isoforms. Further, shifting AS modes may contribute to rapid changes of gene expression during fruit set.© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Read more
September 22, 2019

Cataloguing over-expressed genes in Epstein Barr Virus immortalized lymphoblastoid cell lines through consensus analysis of PacBio transcriptomes corroborates hypomethylation of chromosome 1

The ability of Epstein Barr Virus (EBV) to transform resting cell B-cells into immortalized lymphoblastoid cell lines (LCL) provides a continuous source of peripheral blood lymphocytes that are used to model conditions in which these lymphocytes play a key role. Here, the PacBio generated transcriptome of three LCLs from a parent-daughter trio (SRAid:SRP036136) provided by a previous study [1] were analyzed using a kmer-based version of YeATS (KEATS). The set of over-expressed genes in these cell lines were determined based on a comparison with the PacBio transcriptome of twenty tissues pro- vided by another study (hOPTRS) [2]. MIR155 long non-coding RNA (MIR155HG), Fc fragment of IgE receptor II (FCER2), T-cell leukemia/lymphoma 1A (TCL1A), and germinal center associated signaling and motility (GCSAM) were genes having the highest expression counts in the three LCLs with no expression in hOPTRS. Other over-expressed genes, having low expression in hOPTRS, were membrane spanning 4-domains A1 (MS4A1) and ribosomal protein S2 pseudogene 55 (RPS2P55). While some of these genes are known to be over-expressed in LCLs, this study provides a comprehensive cataloguing of such genes. A recent work involving a patient with EBV-positive large B-cell lymphoma was “unusually lacking various B-cell markers”, but over-expressing CD30 [3] – a gene ranked 79 among uniquely expressed genes here. Hypomethylation of chromosome 1 observed in EBV immortalized LCLs [4, 5] is also corroborated here by mapping the genes to chromosomes. Extending previous work identifying un-annotated genes [6], 80 genes were identified which are expressed in the three LCLs, not in hOPTRS, and missing in the GENCODE, RefSeq and RefSeqGene databases. KEATS introduces a method of determining expression counts based on a partitioning of the known annotated genes, has runtimes of a few hours on a personal workstation and provides detailed reports enabling proper debugging.

Read more
September 22, 2019

Characterization of the Rosellinia necatrix transcriptome and genes related to pathogenesis by single-molecule mRNA sequencing.

White root rot disease, caused by the pathogen Rosellinia necatrix, is one of the world’s most devastating plant fungal diseases and affects several commercially important species of fruit trees and crops. Recent global outbreaks of R. necatrix and advances in molecular techniques have both increased interest in this pathogen. However, the lack of information regarding the genomic structure and transcriptome of R. necatrix has been a barrier to the progress of functional genomic research and the control of this harmful pathogen. Here, we identified 10,616 novel full-length transcripts from the filamentous hyphal tissue of R. necatrix (KACC 40445 strain) using PacBio single-molecule sequencing technology. After annotation of the unigene sets, we selected 14 cell cycle-related genes, which are likely either positively or negatively involved in hyphal growth by cell cycle control. The expression of the selected genes was further compared between two strains that displayed different growth rates on nutritional media. Furthermore, we predicted pathogen-related effector genes and cell wall-degrading enzymes from the annotated gene sets. These results provide the most comprehensive transcriptomal resources for R. necatrix, and could facilitate functional genomics and further analyses of this important phytopathogen.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.