(1119-D) High-quality RNA libraries generated on the Agilent Bravo NGS Workstation with a new, shorter RNA library prep workflow with rRNA depletion

Abstract: Introduction
RNA sequencing (RNA-seq) with ribosomal RNA (rRNA) depletion has become an important, popular tool for studying gene expression. Historically, the preparation of RNA-seq libraries has involved complex workflows with long processing times, and automated methods have required frequent user intervention1. Roche has developed a shorter, time-saving RNA-seq library preparation workflow with its on-market RNA-seq kits to significantly reduce both processing time and hands-on time while maintaining the same high level of performance*. This study demonstrates the successful development of a new automated workflow for this method, further reducing hands-on time and increasing throughput capacity.

Methods
The standard workflow for the KAPA RNA HyperPrep Kit with RiboErase (HMR)
has been shortened by reducing the length of the DNase digestion and second-strand synthesis steps, and by eliminating a bead-based cleanup step. In this study, this shorter workflow was automated on the Agilent Bravo B liquid handler and then sequenced. For comparison, libraries were also prepared using the full-length, standard workflow– both manually and on the Agilent Bravo NGS Workstation

Results
Both pre-sequencing and post-sequencing metrics for all library preparation methods — including total library yield, number of genes and transcripts detected, low duplication rate, and low residual ribosomal RNA alignment — were similar across all library preparation workflows.

Conclusion
The implementation of the shortened workflow for the KAPA RNA HyperPrep Kit with RiboErase (HMR) on the Agilent Bravo NGS Workstation produces results comparable to both manual and automated preparations of the full-length workflow. Thus, this new, faster method produces equivalent RNA-seq libraries in less time while maintaining the lower human error rates and increased throughput expected from an automated method.

Reference:
1. Han Y, Gao S, Muegge K, Zhang W, Zhou B. Advanced Applications of RNA Sequencing and Challenges. Bioinform Biol Insights. 2015 Nov 15;9(Suppl 1):29-46. doi: 10.4137/BBI.S28991. PMID: 26609224; PMCID: PMC4648566.
*Data on file at Roche Diagnostics, Wilmington, MA, USA
For Research Use Only. Not for use in diagnostics procedures. KAPA is a trademark of Roche.

For Research Use Only. Not for use in diagnostics procedures. Bravo is a trademark of Agilent.