(1124-A) Accelerating Discoveries: A Comprehensive RNA-Based Next Generation Sequencing Library Automation Approach

Abstract: With the rise of RNA-based therapeutics, automating methods that validate them is of great importance. RNA-based Next Generation Sequencing (NGS) library preparation assays are an example of these methods. Current RNA NGS methods offer the opportunity to analyze previously extracted Total RNA; Ribosomal RNA (rRNA); Poly (A) RNA; and Formalin-Fixed, Paraffin-Embeded (FFPE) RNA. These assays could be divided into two (2) main parts. The first part is a cDNA Synthesis workflow. This part consists of the fragmentation of the extracted RNA, followed by First Strand Synthesis, and Second Strand Synthesis which ends with a cDNA product. The second part is the Library Preparation Workflow of the obtained cDNA. This part consecutively does the samples' Adenylation, Adapter Ligation, and Primer Amplification. After this last step, the samples are ready to be sequenced and analyzed for their intended application. We have successfully automated a Total RNA-based assay. The protocol was designed for the user to fully walk away from the instrument and would only need to interact with the process when loading Part One (1) and Part Two (2) of the method. Lastly, our final samples are comparable to the manual positive controls of the protocol. We determined this by analyzing their electropherograms and measuring the concentration of all the samples.