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Although RNA-Seq is a relatively new methodology, with some of the first transcriptome-wide analyses appearing around 2008, RNA-Seq has rapidly evolved to become a standard assay for RNA measurement. In the last several years, assays have improved to be high throughput, more specific, work with smaller input amounts, process degraded RNA, and perform well with special tissues (eg, blood samples). On the bioinformatics side, there have been improvements in references, alignment, quantitation, and variant detection methods, and the development of specialized methods, such as transcriptome assembly, fusion discovery, infectious agent detection, and metagenomic expression.
Viewers will learn about the many RNA-Seq related improvements, as well as some of the current limitations in inferences and precision. Some examples of improvements and new capabilities that will be discussed include:
- strand precision
- the ability to measure RNA from a small number of cells
- new protocols for degraded RNA
- and the ability to sequence samples at great depths (up to 1B paired reads) extending the dynamic range to over 6 orders of magnitude.
The presenter will also examine some current limitations including that due to specific biases inherent to each protocol, we cannot make inferences about absolute copy number of a particular RNA species with any precision, only relative copy number, despite the omnipresence of normalization measures such as FPKM that imply otherwise. In addition, until we have technologies that provide full-length sequencing, we will be unable to discern potentially important quantitative and qualitative differences in highly similar isoforms.
Through this webinar viewers will have a much more comprehensive understanding of the types of biological questions and problems RNA-Seq can address fully or be a key aid in addressing, and those it cannot.