Nucleic acids | RNA identification and quantification via next generation sequencing

Detecting and quantifying nucleic acids by RT-qPCR has been the gold standard for decades, but next-generation sequencing (NGS) is now a powerful contender when it comes to unbiased high-throughput profiling experiments. Adopting NGS-based techniques for RNA analysis (RNA-Seq) allows researchers to garner unprecedented insight into the inner workings of transcriptome biology. RNA-Seq facilitates studying a wide variety of RNA species, ranging from full-length, protein-coding mRNAs to small regulatory RNAs such as microRNAs, with single-nucleotide resolution. Despite having progressively established itself as a mainstream tool, RNA-Seq can still seem offputtingly complex to novices in the field. To make it more accessible, this article provides an introduction to sequencing-based analysis of RNA, and details commonly used methods for various research questions. We present major sequencing platforms, including “Nanopore Sequencing,” “Pyrosequencing” and “Illumina sequencing-by-synthesis,” and highlight their respective advantages and pitfalls. These technical discussions are complemented by an introduction to quality control, processing and analysis of RNA-Seq data. Our intention is to provide an entry point into utilizing NGS for transcriptomics research, and to emphasize the advances this promising method looks to bring in the years to come.