Discovering new biology through RNA-Seq

Abstract

Sequencing of RNA (RNA-Seq) was invented approximately 1 decade ago and has since revolutionized biological research. This update provides a brief historic perspective on the development of RNA-Seq and then focuses on the application of RNA-Seq in qualitative and quantitative analyses of transcriptomes. Particular emphasis is given to aspects of data analysis. Since the wet-lab and data analysis aspects of RNA-Seq are still rapidly evolving and novel applications are continuously reported, a printed review will be rapidly outdated and can only serve to provide some examples and general guidelines for planning and conducting RNA-Seq studies. Hence, selected references to frequently update online resources are given.Sequencing of RNA (RNA-Seq) is a recent technique that emerged shortly after next-generation sequencing (NGS) was invented approximately 10 years ago and since has revolutionized biological research in the 21st century. The major advance and basis of NGS is the application of sequencing-by-synthesis technology, which entails real-time monitoring of de novo DNA biosynthesis by imaging methods and reading out the sequence of newly synthesized DNA molecules upon iterative addition of the four different nucleotides. This is in contrast to sequencing after synthesis, which is based on the physical separation of differently sized DNA molecules generated by the chain termination inhibitor method in polyacrylamide gels or by capillary electrophoresis after completion of the sequencing reaction (Sanger et al., 1977).Most of the current sequencing-by-synthesis technologies are based on the immobilization of a denatured, single-stranded sequencing template on a surface, either a glass slide or nano beads. Immobilization on a surface allows for repeated cycles of reagent delivery to the immobilized DNA molecule, which permits solid-phase oligonucleotide primer-initiated synthesis of a new DNA strand, using repetitive and iterative cycles of addition of the nucleotides A, C, G, and T. High-resolution imaging is used to detect the incorporation of the nucleotide, either during or after nucleotide incorporation, followed by iterative additional rounds of nucleotide incorporation. The sequence is then eventually deduced from the imaging data.The first successful NGS approach that gained wide acceptance by the community was 454 sequencing, a massively parallel pyrosequencing approach (Margulies et al., 2005). 454 Sequencing is based on the detection of pyrophosphate released during de novo synthesis of a new DNA strand by DNA polymerase, which allows real-time measurements of DNA biosynthesis (Ronaghi et al., 1998). Pyrophosphate released during DNA synthesis is converted to ATP by the action of sulfurylase, followed by generation of a luminescent light signal from ATP, using firefly luciferase. The major advance in 454 technology was combining pyrosequencing with immobilization of the DNA template to nano beads to allow for solid-phase DNA pyrosequencing. The immobilized DNA template is amplified by emulsion PCR and t...

The Structure and Function of Major Plant Metabolite Modifications

“…Metabolomics is a long way off comprehensivity. While nextgeneration sequencing techniques have rendered transcriptomics approaches very close to providing complete coverage (Weber, 2015) and proteogenomics approaches are also making considerable progress (Zhu et al, 2017), metabolomics lags a long way behind. Indeed, a typical MSbased metabolomics experiment yields more unknown peaks than peaks for which chemical structures can be assigned.…”

Comparative Characterization of the Leaf Tissue of Physalis alkekengi and Physalis peruviana Using RNA-seq and Metabolite Profiling

“…RNA sequencing (RNA-seq) on NGSs can be used for both model and non-model plants (Gongora-Castillo and Buell, 2013; Fukushima and Kusano, 2014; Weber, 2015). Successful transcriptome studies on non-model plants, e.g., crop plants such as the eggplant (Ramesh et al, 2016), pepper (Gordo et al, 2012) and tobacco (Lei et al, 2014) and medicinal plants, e.g., Withania somnifera (Gupta et al, 2013; Senthil et al, 2015) and P. peruviana (Garzon-Martinez et al, 2012) from the Solanaceae family have been published.…”

Genome-Wide Identification, Localization, and Expression Analysis of Proanthocyanidin-Associated Genes in Brassica

“…RNA-seq is widely used to estimate transcript amounts and to obtain a quantitative account of transcript amounts in organisms, organs, tissues, or specific cell types, frequently comparing transcript amounts among different samples (Martin et al, 2013; Weber, 2015). …”