Targeted RNA sequencing for gene discovery and quantification
ABSTRACT: Due to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes, RNA sequencing samples the majority of expressed genes infrequently, resulting in sparse sequencing coverage that can hinder robust isoform assembly and quantification. Targeted RNA sequencing addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for focused sequencing. This enhanced sequencing coverage confers sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of targeted RNA sequencing, from initial probe design considerations, capture of targeted genes, to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than a day, while the central experimental capture stage requires ~7 days. Targetted RNA sequencing of long noncoding RNAs
Project description:Gene splicing requires three basal genetic elements; the 3’ and 5’ splice sites and the branchpoint to which the 5’ intron termini is ligated to form a closed lariat during the splicing reaction. The 5’ and 3’ splice sites that define exon boundaries have been widely identified, revealing pervasive transcription and splicing of human genes. However, the locations of the third requisite element, the branchpoint, are still largely unknown. Here we employ two complementary approaches, targeted RNA sequencing and exoribonuclease digestion, to distil sequenced reads that traverse the lariat junction and, via non-conventional alignment, locate human branchpoint nucleotides. Alignments identify 88,748 branchpoints that correspond to 20% of known introns, with 76% supported by diagnostic sequence mismatch errors. This affords a first genome-wide analysis of branchpoints, describing their distribution, selection, and the existence of a diverse array of overlapping sequence motifs with distinct usage, evolutionary histories, and co-variation with distal splicing elements. The overlap of branchpoints with noncoding human genetic variation also indicates a notable contribution to disease. This annotation and analysis incorporates branchpoints into transcriptomic research and reflects a core role for this element in the regulatory code that governs gene splicing and expression. CaptureSeq identification of branchpoint nucleotides
Project description:We compared the performance of conventional RNAseq with RNA Capture Sequencing (CaptureSeq) to assemble and quantify known RNA spike-Ins and human transcripts. We find CaptureSeq to be superior for the detection and quantification of the 37% lowest expressed genes, and comparable for the next 45% of moderately expressed genes. CaptureSeq contributes only minor technical variation and measures differential gene expression accurately. We demonstrate these advantages by the targeted sequencing of long noncoding RNAs across 20 human tissues, expanding previous annotations two-fold and simultaneously generating a quantitative atlas of expression. This analysis confirms the use of CaptureSeq as an important method for transcriptional profiling. Long noncoding RNA assembly and expression is analysed by targeted RNA sequencing for 20 human tissues and 4 human cell lines
Project description:In this work we aim to improve the understanding of the mouse transcriptome complexity, investigate the expressed fraction of the genome and ameliorate the available mouse annotations. We utilized CatureSeq, a recently described strategy meant to enhance the sequencing coverage of low abundant genes. In our experimental design we generated oligonucleotide probes to select annotated and putative long-noncoding RNA and splice junctions. This allowed us to improve dramatically the sequencing throughput of the targeted regions. As a consequence our approach permitted the simultaneous identification of thousands of exons and the expansion of the already known ones The mouse gene assembly is anlaysed by targeted RNA sequencing of lncRNA and splice junctions. 8 mouse tissues and 16 samples are considered in the analysis. Each sample was added with external RNA controls. The controls are polyadenylated transcripts of known concentration designed to be added to an RNA analysis experiment after sample isolation.
Project description:The aim of the experiment was to gain a higher resolution for specific regions of interest to complement the Hi-C results. Capture-C experiments were performed on four-inducible degrons for Scc1, Ringb, Ring1b-Scc1 and CTCF, and Tir1 control, always in triplicates (three biological replicates per condition).
Project description:Through development of an in vivo orthotopic lung cancer model, we reveal an unanticipated pathway driving spontaneous metastasis that is orchestrated by the developmentally-regulated transcriptional repressor, Capicua (CIC). RNAseq and DNA copy number analysis of H1975 (EGFR-mutant lung adenocarcinoma) cells in the context of drug resistance to erlotinib
Project description:We designed a microarray to test at exonic resolution for genomic imbalance for genes representative of all known chromosomal microdeletion/microduplication syndromes, all known causative genes for ID, all known genes encoding glutamate receptors and their known encoding proteins and all known genes encoding proteins with epigenetic regulatory function. We found 36 de novo copy number variants affecting 35 children in this study. 167 trios, each comprising an affected child and both its normal parents were analyzed by conducting two comparative hybridizations; child vs. mother and child vs. father, and only selecting a CNV if it appeared in both hybridizations, i.e., was de novo. De novo CNVs were independantly validated using quantitative PCR.
Project description:BRAT-associated mRNAs and PUM-associated mRNAs were identified in early Drosophila embryos by RNA co-immunoprecipitation of the endogenous proteins using synthetic antibodies, followed by microarray analysis (RIP-Chip). Nine RNA co-immunoprecipitations were performed. This includes 3 biological replicates each of 1) anti-BRAT RNA co-immunoprecipitations from wild-type 0-3 hour embryos, 2) anti-PUM RNA co-immunoprecipitations from wild-type 0-3 hour embryos, and 3) control antibody RNA co-immunoprecipitations from wild-type 0-3 hour embryos. BRAT samples and PUM samples were each normalized separately with the control samples, for a total of 12 processed samples (3 BRAT with 3 control normalized together, and 3 PUM with 3 control normalized together) from the 9 RNA co-immunoprecipitations.
Project description:Reuse of materials in DNA hybridization based methods has been known since the advent of Southern membranes. Array based comparative genomic hybridization is essentially Southern hybridization with multiple probes immobilized on a solid surface. We have shown that comparative genomic hybridization microarrays fabricated with maskless array synthesizer technology can be used up to four times with application of 1,3-dimethylurea as array-stripping agent. We reproducibly detected chromosomal aberrations, 0.6 to 22.4 Mb in size, in four hybridization rounds using regenerated microarray slides. We have also demonstrated that regenerated arrays can detect smaller alterations, 16 – 200 kbp, such as common copy number variants, as well as complex aberration profiles in tumor. Peripheral blood leukocyte DNA samples from 7 individuals with subchromosomal aberrations vs a pool of female DNA (Promega). The experiments were performed in quadruplicate on succesively regenerated microarrays. One dye swap was performed..
Project description:Investigation of whole genome gene expression level changes in Streptomyces avermitilis delta-aveI mutant, compared to the wild-type strain. The mutants analyzed in this study are further described in Chen L, Lu Y., Chen J, Zhang W, Shu D, Qin Z, Yang S, Jiang W. (2008) Characterization of a negative regulator AveI for avermectin biosynthesis in Streptomyces avermitilis NRRL8165. Appl Microbiol Biotechnol 80(2): 277-86. A six chip study using total RNA recovered from three separate Streptomyces avermitilis NRRL8165 and three separate cultures of a Streptomyces avermitilis NRRL8165 delta-aveI (delta-l) mutant. 3 separate RMA normalizations performed, one for each pair of control and mutant samples.