RNA/cDNA Hybrid Sequencing
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB12643 | ENA |
REPOSITORIES: ENA
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Project description:High-throughput RNA Isoform Sequencing using Programmable cDNA Concatenation
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Project description:We report the complexity of the polyadenylated transcriptome of the mouse intestinal tract assessed by a hybrid RNA-sequencing. We show that 90.72% of the polyA-derived unique reads map to known genes, but 9.28% of reads matched to nonannotated genomic regions. Screening and verification of non-annotated transcriptional units by different RNA-Seq protocols.
2011-01-06 | E-GEOD-21699 | biostudies-arrayexpress
Project description:We applied the solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of 1 million CpGs in more than 21,408 CGIs and 15,946 transcriptional regulatory regions. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. In this study, we generated genome-wide, single-base resolution DNA methylation maps in three of the most commonly used breast cancer cell lines.Differentially methylated regions (DMRs) were identified in the 5?-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X chromosome among the three cell lines. The single CpG resolution methylation maps of many known tumor suppressor genes were also established in the three cell lines. Here we present a novel approach that combines solution-phase hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in targeted CGI and promoter regions. We designed 51,466 single strand DNA oligonucleotides (160-mer) which target 23,441 CGIs and the transcription start sites of 19,369 known genes in the human genome. The synthetic long DNA oligonucleotides were converted into biotinylated RNA probes for solution-phase hybridization capture of target DNA. The captured genomic DNA was treated with sodium bisulfite, amplified by PCR and sequenced using Illumina GA IIx sequencer.
2011-08-01 | E-GEOD-26826 | biostudies-arrayexpress
Project description:Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization.
| S-EPMC4982148 | biostudies-literature
Project description:We report the complexity of the polyadenylated transcriptome of the mouse intestinal tract assessed by a hybrid RNA-sequencing. We show that 90.72% of the polyA-derived unique reads map to known genes, but 9.28% of reads matched to nonannotated genomic regions.
2011-01-06 | GSE21699 | GEO
Project description:Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORF), six novel ORF-containing transcripts, and fifteen transcripts encoding for messages that potentially alter protein functions through truncations or fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Of these, an evolutionary conserved protein was detected containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.
2022-08-30 | PXD034464 | Pride
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of RNA polymerase II phosphorylated at serine 5 (PolII-S5p; the transcription initiation form) in female mouse cultured hybrid cells and female hybrid brain derived from mouse systems with skewed X inactivation based on crosses between C57BL/6J (BL6) and M. spretus. In these systems, alleles can be differentiated by frequent SNPs between mouse species, and the active X (Xa) compared to the haploid set of autosomes from the same species. To examine PolII-S5p occupancy in vivo, ChIP-seq was done in brain from an adult female F1 mouse in which the BL6 X is always active and the spretus X inactive. Uniquely mapped reads containing informative SNPs were assigned to each haploid chromosome set (BL6 or spretus) and were counted to establish allele-specific PolII-S5p occupancy profiles. We found that PolII-S5p allele-specific occupancy with or without normalization by input genomic DNA sequencing data showed that expressed genes on the Xa (>1RPKM) had 30% higher PolII-S5p peak levels at their promoters compared to autosomal genes from the same species (BL6). This result was confirmed by performing an independent allele-specific ChIP-seq analysis on fibroblasts derived from embryonic kidney (Patski cell line) that have the opposite X inactivation pattern from the brain sample, i.e. an Xa from M. spretus and an Xi from BL6. These findings suggest that transcription initiation of X-linked genes is enhanced to contribute to X upregulation in cell lines and in vivo. Examination of allele-specific PolII-S5p occupancy in mouse hybrid cells and brain.
2013-02-15 | E-GEOD-44255 | biostudies-arrayexpress