Whole Transcriptome Sequencing

Overview

Whole transcriptome sequencing comprises the measurement of the complete complement of transcripts in a sample, at a given time. Through interrogation of the whole transcriptome, researchers are able to determine global expression levels of each transcript (both coding and non-coding) and identify exons, introns and their junctions. Analysis of genome-wide differential RNA expression provides researchers with greater insights into biological pathways and molecular mechanisms that regulate cell fate, development, and disease progression. 

Approaches and Techniques

Total RNA library preparation requires the least amount of input material and enables the most comprehensive view of the transcriptome. However, to increase sequencing economy and improve coverage of low-abundance transcripts of interest, undesired content may either be removed prior to library construction or desired content may be selected from total cDNA libraries after library construction. Since the quality of RNA extracted from biological specimens can be highly variable, and quantities limiting, these techniques must be robust, reliable and streamlined to ensure the highest conversion of input into sequenceable material. 

RNA Transcript Depletion

KAPA RNA HyperPrep Kits with RiboErase (HMR) or RiboErase (HMR) Globin provide for the selective depletion of transcripts (e.g., rRNA, globin mRNA and/or other highly abundant mRNA species), followed by cDNA synthesis and library construction. The enzymatic-depletion chemistry is robust, compatible with both high-quality and degraded inputs (such as FFPE) from all organisms. 

Depletion workflows provide a complete view of the transcriptome, while minimizing the number of wasted sequencing reads from “uninteresting” transcripts. 

Targeted Sequencing

Total RNA inputs, from variable quality samples, are converted into cDNA library molecules using KAPA RNA HyperPrep Kits. This is followed by hybridization capture of targets of interest, which requires only a fraction of the read depth of total RNA sequencing. Depending on the design of the capture panel, this approach allows for interrogation of coding content only (e.g., when using an exome panel), or both and non-coding regions. Since it does not rely on intact 3'-poly(A) tails, this method is compatible with RNA of any quality, and from all organisms.