Project description:In this project we explore the cellular heterogeneity of a mouse model of heart failure with preserved ejection fraction (HFpEF) involving a two-hit model of feeding a high fat diet (HFD) along with L-NAME administration. Healthy adult male mice (C57BL/6J inbred) were fed either a normal chow diet or HFD/L-NAME for 10 weeks or 15 weeks before performing sequencing experiments. Both cardiomyocytes (CMs) and total interstitial population (TIP) were captured using a protocol to jointly capture and sequence single-nuclei (for cardiomyocytes) and single-cells (for TIP) using the 10x Genomics Chromium system.
Project description:To determine if random biopsies can be safely eliminated from screening of average risk persons with IBD, the investigators propose to carry out a pilot randomized control trial in which targeted biopsies in combination with random biopsies will be compared to targeted biopsies alone in terms of pre-cancerous lesion capture rate, side-effects and CRC risk. The pilot study will aim to capture 20% of the overall study population in order to evaluate the feasibility of recruiting the needed number of participants in the specified time frame, while maintaining high quality of data collection.
Project description:To test if scRNA-seq contains sufficient phylogenetic information to reconstruct a population history of cancer, immunosuppressed NU/J mice were injected with human cancer cells (MDA-MB-231-LM2). The tumors that develop are derived from the same population and thus share a common ancestor, but evolved independently in each mouse and should form separate clades on reconstructed phylogenetic trees when analysed together. We explore and compare results of phylogenetic analyses based on both expression levels and SNVs called from our scRNA-seq data. Both techniques are shown to be useful for reconstructing phylogenetic relationships between cells, refecting the clonal composition of a tumor. Without an explicit error model, standardized expression values appears to be more powerful and informative than the SNV values at a lower computational cost, due to being a by-product of standard expression analysis. Our results suggest that scRNA-seq can be a competitive alternative or useful addition to conventional scDNA-seq phylogenetic reconstruction. Our results open up a new direction of somatic phylogenetics based on scRNA-seq data. Further research is required to refne and improve these approaches to capture the full picture of somatic evolutionary dynamics in cancer.
Project description:Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12. We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas. Here, using phylogenetics, structural predictions, comparative genomics, and functional assays, we uncover multiple instances of programmable transcription factors that we name TnpB-like nuclease-dead repressors (TldR). These proteins employ naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPRi technologies invented by humans. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility, phage susceptibility, and host immunity. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.
Project description:Accurate annotations of genes and their transcripts is a foundation of genomics, but no annotation technique presently combines throughput and accuracy. As a result, the GENCODE reference collection of long noncoding RNAs remains far from complete: many are fragmentary, while thousands more remain uncatalogued. To accelerate lncRNA annotation, we have developed RNA Capture Long Seq (CLS), combining targeted RNA capture with third generation long-read sequencing. We present an experimental re-annotation of the entire GENCODE intergenic lncRNA populations in matched human and mouse tissues. CLS approximately doubles the complexity of targeted loci, both in terms of validated splice junctions and transcript models. Through its identification of full-length transcript models, CLS allows the first definitive measurement of promoter features, gene structure and protein-coding potential of lncRNAs. Thus CLS removes a longstanding bottleneck of transcriptome annotation, generating manual-quality full-length transcript models at high-throughput scales.
Project description:The leaf transcriptome of the nickel hyperaccumulator species Homalium kanaliense (Salicaceae) endemic from New caledonia were compared to the closely related non-accumulator Homalium betulifolium, living on Gallery forest or maquis on serpentine soil, to identity differentially expressed genes potentially involved in Ni hyperaccumulation.
Project description:We provide raw gene sequences of 174 flowering time regulatory genes and gene othologs across a large barley population (895 barley lines) selected from a collection of landrace, cultivated barley, and research varieties of diverse origin. This set represents the whole variety of cultivated barley lifeforms, namely two- and six-row genotypes with winter, spring, and facultative growth habits. We applied a target capture method based on in-solution hybridization using the myBaits® technology (Arbor Biosciences, Ann Arbour, MI, USA) which is based on in-solution biotinylated RNA probes. Baits were designed for flowering time regulatory genes and gene othologs, and used for production of 80mer capture oligonucleotides for hybridization. Genomic DNA was extracted from leaves of a single two-week old barley plant per variety using the cetyl-trimethyl-ammonium bromide (CTAB) method. Physical shearing of genomic DNA was performed with an average size of 275 bp. Library preparation was conducted with KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA). Hybridization of customised RNA baits with capture pools was performed at 65°C for 24 hours. Each pooled sequence capture library was sequenced on an Illumina HiSeq3000 instrument using three lanes to generate paired-end reads per sample. Genome sequencing was conducted at AgriBio, (Centre for AgriBioscience, Bundoora, VIC, Australia).