Clinical OMICS

JUL-AUG 2019

Healthcare magazine for research scientists, labs, pathologists, hospitals, cancer centers, physicians and biopharma companies providing news articles, expert interviews and videos about molecular diagnostics in precision medicine

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Page 30 of 51 July/August 2019 Clinical OMICs 29 With the increased sequencing throughput and cost reductions, long-read full transcriptome sequencing is now being performed, either to generate highly accurate annotations for newly assembled genomes, to further expand and improve existing annotations, or conduct differential splicing analyses. Alternative splicing has been well described to be regulated across development, tissue, and cell types. With the rise of single-cell sequencing, long-read sequencing is now being applied to identify cell-specific splicing patterns. Current single-cell sequencing approaches relying on 3' end sequence, while enabling the characterization of the transcriptome of thousands of single cells in a single experiment, prevent transcript level analysis. The first studies that applied long-read sequencing made use of PCR amplification to reach the required input. However, new protocols take advantage of microfluidic approaches to isolate cells and barcode the endogenous RNA prior to pooling for long-read sequencing, relying on computational identification of cells. This approach allows the screening of thousands of cells, and despite the relatively low read count per cell, has already enabled the annotation of 10,691 novel transcripts affecting 4,859 genes and the identification of cell type-specific splicing. 5 With the increased sequencing output of new platforms and lower input requirements, single-cell full transcriptomes will soon be attained. The democratization of long-read technologies means that novel applications pertaining to RNA are appearing. RNA modifications 6 as well as RNA secondary structure can now be probed through direct RNA sequencing. This revolution in RNA sequencing technologies opens new areas of investigation in biology. Picture: Plenary speaker Nicola Hall, University of Oxford, who presented on revealing mRNA alternative splicing complexity in the human brain. REFERENCES: 1. Steijger, T. et al. Assessment of transcript reconstruction methods for RNA-seq. Nat Methods. 10:1177–1184 (2013). 2. Sun, W. et al. Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing. EMBO J. 32(14):2029–2038 (2013). 3. Clark, M. et al. Long-read sequencing reveals the splicing profile of the calcium channel gene CACNA1C in human brain. BioRxiv. 260562 (2018). 4. Treutlein, B., Gokce, O., Quake, S.R., and Südhof, T.C. Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing. PNAS. 111(13):E1291– E1299 (2014). 5. Gupta, I. et al. Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells. Nat Biotechnol. 36(12):1197–1202 (2018). 6. Wongsurawat, T. et al. Rapid Sequencing of Multiple RNA Viruses in Their Native Form. Front Microbiol 10:260 (2019). July/August 2019 Clinical OMICs 29

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