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

Abstract

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.

De novo assembly of the olive fruit fly (Bactrocera oleae) genome with linked-reads and long-read technologies minimizes gaps and provides exceptional Y chromosome assembly

“…Further, we were able to extract high molecular weight DNA that was used to prepare long-read and linked-read sequencing libraries. Due to technological advancements, the library preparation and sequencing costs of short-and long-read technologies is converging at USD 20-40 per Gb [98]. Linked-reads library was calculated for each of the stages; egg, larvae, pupae, and adult using RSEM [94].…”

De novogenome assembly of the olive fruit fly (Bactrocera oleae) developed through a combination of linked-reads and long-read technologies

“…Unfortunately, due to the short-read nature of this technology de novo genome assemblies for even prokaryotes are per Gb, which is ~5-10 times more than Illumina 41 . However, the ONT PromethION can cost USD 12 -24 per Gb due to increased throughput 41 . In parallel, long-read sequencing library preparation protocols put a considerable demand on DNA quantity required for constructing and sequencing long-read libraries 42 .…”

Introductory Chapter: Transcriptome Analysis

“…Nowadays the microarray approach is supplanted by high-throughput RNA sequencing, RNA-Seq, which detects all transcripts in a sample, including the regulatory siRNA and lncRNA transcripts [3]. In this methodology, the bulk RNA is extracted from the sample and copied into stable double-stranded copy DNA, ds-cDNA, which is then sequenced using various sequencing methods [4].…”