Advanced Applications of RNA Sequencing and Challenges

Abstract

Next-generation sequencing technologies have revolutionarily advanced sequence-based research with the advantages of high-throughput, high-sensitivity, and high-speed. RNA-seq is now being used widely for uncovering multiple facets of transcriptome to facilitate the biological applications. However, the large-scale data analyses associated with RNA-seq harbors challenges. In this study, we present a detailed overview of the applications of this technology and the challenges that need to be addressed, including data preprocessing, differential gene expression analysis, alternative splicing analysis, variants detection and allele-specific expression, pathway analysis, co-expression network analysis, and applications combining various experimental procedures beyond the achievements that have been made. Specifically, we discuss essential principles of computational methods that are required to meet the key challenges of the RNA-seq data analyses, development of various bioinformatics tools, challenges associated with the RNA-seq applications, and examples that represent the advances made so far in the characterization of the transcriptome.

Keywords: RNA-seq; alternative splicing; co-expression network; data preprocessing; differential gene expression; pathway analysis; systems biology; variants detection.

Figure 1

Overview of the typical RNA-seq pipeline. Three main sections are presented: The Experimental Biology, The Computational Biology and The Systems Biology. The pipeline starts from the experimental preparation and come with the work flow to the sequencing and analysis steps as the arrows point from step to step.

Figure 2

The STARR-seq pipeline and the corresponding ‘systems biology’ steps. The sonicated genomic DNA are PCR amplified and placed downstream of a minimal promoter in reporter vectors. The desired measurement are embedded in the genome. The reporter library is transfected into the cultured cell lines and Poly-A RNAs are isolated from the pool of total RNA. These steps are selectively to enrich the targets interested. After RNA-seq is performed, the reads are mapped to the reference genome and their enrichment over input are measured to reflect enhancer activity. The steps of systems biology including mathematics and computational biology analysis will help with the interpretation.