Project description:Here, we sequenced and functionally annotated the long reads (1-2 kb) cDNAs library of an infratentorial ependymoma tumor tissue on PacBio RSII by Iso-Seq protocol using SMRT technology. 577 MB, data was generated from the brain tissues of ependymoma tumor patient, producing 1,19,313 high-quality reads assembled into 19,878 contigs using Celera assembler followed by Quiver pipelines, which produced 2952 unique protein accessions in the nr protein database and 307 KEGG pathways. Additionally, when we compared GO terms of second and third level with alternative splicing data obtained through HTA Array2.0. We identified four and twelve transcript cluster IDs in Level-2 and Level-3 scores respectively with alternative splicing index predicting mainly the major pathways of hallmarks of cancer. Out of these transcript cluster IDs only transcript cluster IDs of gene PNMT, SNN and LAMB1 showed Reads Per Kilobase of exon model per Million mapped reads (RPKM) values at gene-level expression (GE) and transcript-level (TE) track. Most importantly, brain-specific genes--PNMT, SNN and LAMB1 show their involvement in Ependymoma.
Project description:We investigated cross-talk between the membrane-associated, myosin II-regulatory protein supervillin and the actin-regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1-GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLa cells with normal levels of the Rac1-activating protein Trio. No reduction in Rac1-GTP loading was observed when supervillin levels were reduced in Trio-depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio-mediated Rac1 guanine nucleotide exchange activity with ITX3 partially blocked the SV4-mediated increase in Rac1-GTP. Both SV4 and SV1 co-localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4-7: SV1-171, which contains N-terminal residues found in both SV1 and SV4 and the SV4-specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4-E345; SV4 amino acids 276-669). In addition, SV4-E345 interacted with the homologous sequence in rat kalirin (repeats 4-7, amino acids 531-1101). Overexpressed SV1-174 and SV4-E345 affected Rac1-GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771-1057, which contain both supervillin-interaction sites, exerted a dominant-negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension.
Project description:We used PacBio data to identify more reliable transcripts from hESC, based on which we can estimate gene/transcript abundance better from Illumina data. PacBio long reads and Illumina short reads were generated from the same hESC cell line H1. PacBio reads were error-corrected by Illumina reads to identify transcripts. rSeq is used to estimate gene/transcript abundance of the identified transcriptome.
Project description:Heterozygous coding mutations in TRIO are associated with neurodevelopmental disorders, including autism, schizophrenia, bipolar disorder, and epilepsy, and impair TRIO's biochemical activities. To model mutant alleles, we ablated one or both Trio alleles from excitatory neurons in the cortex and hippocampus of mice. Trio haploinsufficiency increases anxiety and impairs social preference and motor coordination. Trio loss reduces forebrain size and dendritic arborization but increases dendritic spine densities. Cortical synapses in Trio haploinsufficient mice are small, exhibit pre- and postsynaptic deficits, and cannot undergo long-term potentiation. Similar phenotypes are observed in Trio knockout mice. Overall, Trio haploinsufficiency causes severe disease-relevant deficits in behavior and neuronal structure and function. Interestingly, phosphodiesterase 4A5 (PDE4A5) levels are reduced and protein kinase A (PKA) signaling is increased when TRIO levels are reduced. Elevation of PDE4A5 and drug-based attenuation of PKA signaling rescue Trio haploinsufficiency-related dendritic spine defects, suggesting an avenue for therapeutic intervention for TRIO-related neurodevelopmental disorders.
Project description:Case-parent trio studies are commonly employed in genetics to detect variants underlying common complex disease risk. Both commercial and freely available software suites for genetic data analysis usually contain methods for case-parent trio designs. A user might, however, experience limitations with these packages, which can include missing functionality to extend the software if a desired analysis has not been implemented, and the inability to programmatically capture all the software versions used for low-level processing and high-level inference of genomic data, a critical consideration in particular for high-throughput experiments. Here, we present a software vignette (i.e., a manual with step by step instructions and examples to demonstrate software functionality) for reproducible genome-wide analyses of case-parent trio data using the open source Bioconductor package trio. The workflow for the practitioner uses data from previous genetic trio studies to illustrate functions for marginal association tests, assessment of parent-of-origin effects, power and sample size calculations, and functions to detect gene-gene and gene-environment interactions associated with disease.
Project description:The detection and identification of species of fungi in the environment using molecular methods heavily depends on reliable reference sequence databases. However, these databases are largely incomplete in terms of taxon coverage, and a significant effort is required from herbaria and living fungal collections for the mass-barcoding of well-identified and well-curated fungal specimens or strains. Here, a PacBio amplicon sequencing approach is applied to recent lichen herbarium specimens for the sequencing of the fungal ITS barcode, allowing a higher throughput sample processing than Sanger sequencing, which often required the use of cloning. Out of 96 multiplexed samples, a full-length ITS sequence of the target lichenised fungal species was recovered for 85 specimens. In addition, sequences obtained for co-amplified fungi gave an interesting insight into the diversity of endolichenic fungi. Challenges encountered at both the laboratory and bioinformatic stages are discussed, and cost and quality are compared with Sanger sequencing. With increasing data output and reducing sequencing cost, PacBio amplicon sequencing is seen as a promising approach for the generation of reference sequences for lichenised fungi as well as the characterisation of lichen-associated fungal communities.
Project description:Nascent transcriptome sequencing (GRO-seq) experiment of 3 human lymphoblastoid cell line samples from the 1000 Genomes sample set (http://www.1000genomes.org/). Dataset includes one parent-daughter trio (CEU populations). This accession contains raw, mapped, and processed GRO-seq read data, other assays in this study are available under accession E-MTAB-1884 (ChIP-seq, https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1884) and E-MTAB-1883 (RNA-seq, https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1883/).