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Transcript Profiling Using Single Molecule, Long-Read Sequencing Technologies

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RNA sequencing using next generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large scale high-throughput studies. NGS technologies comprise of high throughput, cost efficient short-read RNA-Seq while new, emerging single molecule, ...

RNA sequencing using next generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large scale high-throughput studies. NGS technologies comprise of high throughput, cost efficient short-read RNA-Seq while new, emerging single molecule, long-read RNA-Seq technologies have opened up new and rapidly changing possibilities to study the transcriptome and its function. These emerging single molecule, long read technologies are currently by Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT), while new methodologies based on short read sequencing approaches are being developed in order to provide long range single molecule level information, for example represented by 10X Genomics.

The shift towards long-read sequencing technologies for transcriptome characterization is based on current increases in throughput and decreases in cost, making these attractive for de novo transcriptome assembly, isoform expression quantification and in-depth RNA species analysis. These types of analyses were challenging with standard short sequencing approaches due to the complex nature of the transcriptome which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes as well as the high sequence similarity of highly abundant species of RNA, such as rRNAs.

Here we aim to focus on single molecule level sequencing technologies and single cell technologies, which, combined with perturbation tools, allow the analysis of complete RNA species, whether short or long at high resolution. In parallel these tools have opened new ways in understanding gene functions at the tissue, network and pathway level, as well as their detailed functional characterisation.

Analysis of the epi-transcriptome, including RNA methylation and modification and the effects of such modifications on biological systems. Direct RNA sequencing is a growing field and will also make RNA modifications easier to detect and quantify. However, many difficulties and challenges remain, such as methodologies to generate full length RNA or cDNA libraries from all different species of RNAs, not only polyA containing transcripts, the identification of allele specific transcripts due to current error rates of single molecule technologies, while the bioinformatics analysis on ling read data for accurate identification of 5’ and 3’UTRs is still in development.

Manuscripts we would like to be submitted include:

1. Methodological developments for full length cDNA library preparation
2. Methodological developments for sequencing native RNA
3. Application of long read technologies in de novo characterization of RNA species
4. Application of single molecule, long read sequencing in de novo characterization of transcriptomes of different organisms, tissues or cell types
5. Data analysis approaches for the identification of 5’ and 3’ UTRs from long read RNA-seq data
6. Epitranscriptome analysis, including RNA methylation and base modification through the application of long read/single molecule technologies
7. Methodologies for RNA isoform identification
8. Sequencing technology developments allowing full RNA species characterization or epitranscriptomics
9. Methodological and analytical approaches for allele specific transcript identification using long read/single molecule technologies.


Keywords: RNA-Seq, Long read, Pacbio, Nanopore, Next Generation Sequencing, Transcriptome


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