Current and Future Methods for mRNA Analysis: A Drive Toward Single Molecule Sequencing

The transcriptome encompasses a range of species including messenger RNA, and other noncoding RNA such as rRNA, tRNA, and short and long noncoding RNAs. Due to the huge role played by mRNA in development and disease, several methods have been developed to sequence and characterize mRNA, with RNA sequencing (RNA-Seq) emerging as the current method of choice particularly for large high-throughput studies. Short-read RNA-Seq which involves sequencing of short cDNA fragments and computationally assembling them to reconstruct the transcriptome, or aligning them to a reference is the most widely used approach. However, due to inherent limitations of this approach in de novo transcriptome assembly and isoform quantification, long-read RNA-Seq approaches, which also happen to be single molecule sequencing approaches, are increasingly becoming the standard for de novo transcriptome assembly and isoform quantification. In this chapter, we review the technical aspects of the current methods of RNA-Seq, both short and long-read approaches, and data analysis methods available. We discuss recent advances in single-cell RNA-Seq and direct RNA-Seq approaches, which perhaps will dominate the future of RNA-Seq.