Effects of Pseudorabies Virus Infection on the Tracheobronchial Lymph Node Transcriptome
ABSTRACT: RNA isolated from draining tracheobronchial lymph nodes (TBLN) from 5-week old pigs, either clinically infected with a feral isolate of Pseudorabies virus or uninfected were interrogated using Illumina Digital Gene Expression Tag Profiling. Over 100 million tag sequences were observed, representing 4,064,189 unique 21-base sequences collected from TBLN at time points 1, 3, 6 and 14 days post-infection (dpi) RNA isolated from draining tracheobronchial lymph nodes (TBLN) from 5-week old pigs (% per group pooled), either clinically infected with feral isolate FS268 of Pseudorabies virus or uninfected at 1, 3, 6, and 14 days post inoculation. Over 100 million tag sequences were observed, representing 4,064,189 unique 21-base sequences.
Project description:Background: A comprehensive transcriptome survey, or "gene atlas", provides information essential for a complete understanding of the genomic biology of an organism. We present an atlas of RNA abundance for 92 adult, juvenile and fetal cattle tissues and 3 cattle cell lines. Results: The Bovine Gene Atlas was generated from 7.2 million unique digital gene expression tag sequences (300.2 million total raw tag sequences), from which 1.59 million unique tag sequences were identified that mapped to the bovine genome accounting for 85% of the total raw tag abundance. Filtering these tags yielded 87,764 unique tag sequences that unambiguously mapped to 16,517 annotated protein-coding loci in the genome accounting for 45% of the total raw tag abundance. Clustering of tissues based on tag abundance profiles generally confirmed ontology classification based on anatomy. There were 5,429 constitutively expressed loci and 3,445 constitutively expressed unique tag sequences mapping outside annotated gene boundaries that represent a resource for enhancing current gene models. Physical measures such as inferred transcript length or antisense tag abundance identified tissues with atypical transcriptional tag profiles. We report for the first time the tissue specific variation in the proportion of mitochondrial transcriptional tag abundance. The Bovine Gene Atlas can be examined at http://www.agbase.msstate.edu/bovineatlas . Conclusions: The Bovine Gene Atlas is the deepest and broadest transcriptome survey of any livestock genome to date. Commonalities and variation in sense and antisense transcript tag profiles identified in different tissues facilitate the examination of the relationship between gene expression, tissue, and gene function. An atlas of mRNA abundance for 92 adult, juvenile and fetal cattle tissues and 3 cattle cell lines.
Project description:Schistosoma japonicum is one of the remarkable platyhelminths that are endemic in the South Asian countries. The parasite is dioecious and can parasitize inside host blood stream for many years. Rapid reproduction by producing large number of eggs and count-react or active manipulating host anti-parasite responses are the strategies that benefit the long term parasitization of the parasite. Currently, Praziquantel is the only commercial drug that is effective against the worms but not the eggs of parasite in the host and no vaccine available to prevent human or animal schistosomiasis. Development of novel antiparasite reagents and immune-prevention measures rely on the deciphering of parasite biology. The decoding of the genomic sequence of the parasite has made it possible to dissect the biological functions of genes that govern the development of the parasite inside the hosts. In this study, the polyadenylated RNA from male and female S. japonicum was isolated and deep sequenced. By comparative analysis on the transcriptomic differences of the two sexes of the parasite, potential genes or gender-specific biological pathways can be targeted. Messenger RNA from male and female S. japonicum parasite was selectively purified from total RNA using oligo-(dT) conjugated magnetic beads. Complementary DNA (cDNA) was synthesized guided by oligo-(dT) as a primer.
Project description:Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression (DGE) profiles using Illumina’s high-throughput sequencing technology and the newly-assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in determinacy of axillary meristems. The RA3 gene encodes a trehalose-6-phosphate-phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of DGE libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20-21nt reads with frequencies spanning four orders of magnitude. Unique sequence tags were anchored to 3´-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of non-redundant signature tags to the maize genome, which associated with 37,117 working gene models and un-annotated regions of expression. 66% of maize genes were detected at ≥ nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis and rice in functional analyses of differentially expressed maize genes. Results from this study provide a basis for analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene. 3' tag-based DGE libraries were generated using Illumina 1G technology. Libraries were prepared from 2mm ear samples and represent: 3 biological replicates of wild-type B73, 3 biological replicates of ramosa3 (ra3) mutants, and 1 technical replicate of a ra3 sample. Each sample was sequenced in a single lane of a flow cell. Each lane includes library preparations from both DpnII and NlaIII digests for that sample. Sequences from DpnII digests start with GATC and those from NlaIII digests start with ATG.
Project description:Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethal infection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptome level, we have extended and validated previous microarray data by Illumina next-generation sequencing analysis. Comparison of tag-based sequencing (DGE or Tag-Seq) with full transcript sequencing (RNA-Seq) showed a strong correlation of sequence read counts per transcript and an overlap of 241 transcripts differentially expressed in response to infection. A slightly lower overlap of 165 transcripts was observed with previous microarray data. Based on the combined sequencing-based and microarray-based transcriptome data we compiled an annotated reference set of infection-responsive genes in zebrafish embryos, encoding transcription factors, signal transduction proteins, cytokines and chemokines, complement factors, proteins involved in apoptosis and proteolysis, proteins with anti-microbial activities, as well as many known or novel proteins not previously linked to the immune response. Furthermore, by comparison of the deep sequencing data of S. typhimurium infection in zebrafish embryos with previous deep sequencing data of Mycobacterium marinum infection in adult zebrafish we derived a common set of innate host defense genes that are expressed both in the absence and presence of a fully developed adaptive immune system and that provide a valuable reference for future studies of host-pathogen interactions using zebrafish infection models. Zebrafish embryos were infected with Salmonella typhimurium (strain SL1027) by microinjection of DsRED-labeled bacteria into the caudal vein close to the urogenital opening after the onset of blood circulation (27 hpf). An equal volume of PBS was injected in the control group. RNA samples were collected at 8 hours post infection (hpi) and samples from triplicate infection experiments were pooled. DGE libraries from the RNA pools (1 μg) of Salmonella-infected and control embryos were prepared using the DGE:Tag Profiling for NlaIII Sample Prep kit from Illumina as previously described (Hegedus et al., 2009). The libraries were sequenced in duplicate using 2 (Control 1; Salmonella infected 1) and 3 pmol (Control 2; Salmonella infected 2) of cDNA. Sequencing was performed using the Illumina Genome Analyzer II System (BaseClear B.V., Leiden, The Netherlands) according to the manufacturer’s protocols. Image analysis, base calling, extraction of 17 bp tags and tag counting were performed using the Illumina pipeline. Tag counts from duplicate libraries were merged in silico.
Project description:We used a digital gene expression (DGE) system to generate partial gene expression profiles for 12 selected samples. Significant differences in gene expression between early- and late-flowering samples were detected for 72 candidate genes for flowering time. Genes related to circadian rhythms were significantly overrepresented among the differentially expressed genes. Our data suggest that circadian clock genes play an important role in the evolution of flowering time, and C. bursa-pastoris plants exhibit expression differences for candidate genes likely to affect flowering time across the broad range of environments they face in China. Digital gene expression analysis was performed on RNA from 12 different flowering samples of C. bursa-pastoris. These samples are from different areas, 6 of which represent early-flowering ecotypes, and the other 6 samples represent late-flowering samples
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of transcriptome in mazie plants. The ZmPIS gene coding PtdIns synthase from maize with a maize ubiquitin promoter was transferred into maize. The transgenic ZmPIS maize showed enhanced drought tolerance compared to non-transgenic maize. The differentially expressed genes between wide-type maize and transgenic ZmPIS maize were detected by the assay of digital gene expression profile and real time RT-PCR datas. The results displayed that the overexpression of ZmPIS resulted in the expression levels changes of a large number of genes including genes involved in the phosphatidylinositol (PI) metabolic pathway, photosynthesis metabolism, carbohydrate metabolism, aminoacid metabolism and genes coding transcription factors. Examination of The differences of the transcriptional profile between wide-type maize and transgenic ZmPIS maize and analysis of the network regulated by the ZmPIS gene
Project description:The goals of this study are to compare differential gene expressions for Penicillium oxalicum wild type strain (WT), and laeA knockout strain (ΔlaeA) in different development phase. The deletion of laeA downregulated genes involved in oxidation- reduction process, alkaloid metabolic process, and transmembrane transport. We find the expression levels of seven secondary metabolism gene clusters (totally 28 clusters) were silenced inΔlaeA. This study provides the information that laeA function are required in conidiation and hydrolase activity of P. oxalicum. Examination of differential gene expressions by digital gene expression tag profiling in Penicillium oxalicum wild type strain and laeA knockout mutant strains in 24h and 60h in modified Czapek culture medium with 2% glucose as carbon resource. qRT–PCR validation was performed using SYBR Green assays.
Project description:Obese and lean pig show breed-specific traits in muscle growth and meat quality. However, the mechanisms underlying remains unclear. Here, we reported the first genome-wide muscle development research from embryonic to 6 months old between Lantang (obese) and Landrace (lean) using Solexa/Illumina’s Genome Analyzer. We obtained 4.22±0.9×107 total reads per sample with approximately 5×106 distinct tags. Filtered adaptor tags, empty reads, low quality tags and tags of copy number =1, we obtained 3.84±0.9×107 clean total tags with 1.5±0.5×106 clean distinct tags. 20 libraries of longissimus muscle samples were sequenced in Lantang and Landrace at days 35, 49, 63, 77, 91 prenatal and 2, 28, 90, 120, 180 postnatal days.
Project description:Transcriptomic analysis of fungus Penicillium decumbens and brlA deletion strains in liquid medium and solid medium respectivelly Examination of differential gene expressions by Penicillium decumbens strains 114-2 and brlA deletion stains in liquid medium and solid medium
Project description:Trichomes are the hair-like structures that are widely present on the surface of aerial organs and function in plant defense against biotic and abiotic stresses. Previous studies focus on the single cell trichomes in Arabidopsis and cotton, or multicellular glandular trichomes in tomato, but the developmental process and molecular mechanisms controlling multicellular non-glandular trichome development are largely neglected. Here, we extensively characterized the fruit trichome (spine) development in wild type cucumber and in a tiny branched hair (tbh) mutant that contains a spontaneous mutation and has hairless foliage and smooth fruit surface. Our data indicated that cucumber trichome was multicellular and non-glandular, with no branches or endoreduplication. Further, the major feature of cucumber trichome development was spine base expansion. Transcriptome profiling through Digital Gene Expression indicated that meristem-related genes and transcription factors were implicated in the fruit spine development, and polarity regulators were upregulated during spine base expansion. qRT-PCR verified the reliability of our RNA-SEQ data, and in situ hybridization confirmed the enriched expression of meristem regulators CUP-SHAPED COTYLEDON3 (CUC3) and STM (SHOOT MERISTEMLESS) , as well as the abaxial identity gene KANADI (KAN) in cucumber fruit spine. Together, our results suggest a distinct regulatory pathway involving meristem genes and polarity regulators in multicellular trichome development in cucumber. Using Digital Gene Expression technology to compare the genome-wide gene expression profiles in the fruit spines of wild type cucumber and the tbh mutant, as well as the fruit spines on fruits of 0.5cm and 1.6cm long, repectively. Two biological repelicates were generated for each tissue.