Project description:To identify full-length cap-to-poly(A) mRNA isoforms of CD20 and rule out reverse transcription artifacts which are common in cDNA-seq approaches, long-read Oxford Nanopore direct RNA sequencing was performed on the Raji cell line.
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.
Project description:Ongoing improvements to next generation sequencing technologies are leading to longer sequencing read lengths, but a thorough understanding of the impact of longer reads on RNA sequencing analyses is lacking. To address this issue, we generated and compared two RNA sequencing datasets of differing read lengths -- 2x75 bp (L75) and 2x262 bp (L262) -- and investigated the impact of read length on various aspects of analysis, including the performance of currently available read-mapping tools, gene and transcript quantification, and detection of allele-specific expression patterns. Our results indicate that, while the scalability of read-mapping tools and the cost-effectiveness of long read protocol is an issue that requires further attention, longer reads enable more accurate quantification of diverse aspects of gene expression, including individual-specific patterns of allele-specific expression and alternative splicing. Two RNA-Seq datasets of differing read lengths (2x262 bp and 2x75 bp)
Project description:Evaluation of short-read-only, long-read-only, and hybrid assembly approaches on metagenomic samples demonstrating how they affect gene and protein prediction which is relevant for downstream functional analyses. For a human gut microbiome sample, we use complementary metatranscriptomic, and metaproteomic data to evaluate the metagenomic-based protein predictions.
Project description:Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. To date, long-read RNA sequencing has not been applied to kidney cancer. Here, we used ONT long-read Direct RNA sequencing to profile the transcriptomes of ccRCC cell line RCC4, with and without exposure to pro-inflammatory cytokines. Our results revealed differentially expressed genes induced by the pro-inflammatory cytokines. Moreover, results here revealed potential tumour origin of novel isoforms and genes that were discovered in the archival tumour samples by long-read sequencing.
Project description:Long read SMRT cDNA sequencing of nascent RNA from exponentially growing S. cerevisiae and S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3â?? end was labeled with a 3â?? DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a PCR (gene-specific or transcriptome-wide). SMRT DNA sequencing libraries were prepared subsequently. Nascent RNA profiles for mainly intron-containing genes were generated with long-read SMRT cDNA sequencing.