Abstract 764: Automated walk away NGS sample preparation for constructing in-line molecular barcoded RNA libraries from Fresh Frozen and FFPE samples

Abstract

Abstract RNA-Seq is a revolutionary technology for transcriptome analysis, which has been used to quantify transcript levels, confirm gene annotation, identify novel transcripts, splice variants, and fusion detection. Traditionally, the usefulness of RNA-Seq has been limited by pre-analytical (sample availability, low integrity, tumor heterogeneity, complex protocols) and analytical factors (small mRNA fraction, high read duplicate rate), which can lead to higher costs, longer turnaround times, and false negative results. We have developed and automated a streamlined workflow for construction of highly complex RNA Libraries from fresh frozen and FFPE samples that addresses these concerns. We use target enrichment by bait selection with FFPE-derived samples, where rRNA depletion is found to be inconsistent and poly(A) enrichment cannot be used due to degradation of the source material. A fully automated protocol using pre-aliquoted reagents, has been optimized on the Magnis NGS Prep System for a wide range of RNA inputs (10-200 ng), various sample types (Intact and FFPE total RNA) and equipped with 192 Unique Dual sample Indices (UDI) to minimize the effects of possible index hopping and contamination. Addition of in-line molecular barcodes incorporated at the ligation step helps differentiate fragmentation from PCR duplicates, which increases the total number of reads available for further analysis (gene expression and fusion detection). With minimal risk of contamination, this automated protocol delivers up to eight target-enriched NGS Illumina sequencing-ready cDNA libraries from total RNA input per run in about 12 hours without the need of any user intervention. Both catalog and custom probes can be utilized, and PCR cycles can easily be adjusted from the touchscreen to optimize the library yields. For Research Use Only. Not for use in Diagnostic procedures. Citation Format: Saharnaz Bigdeli, Bahram Arezi, Jan Godoski, Denise Rhodes, Ryan Aguilar, Karen William, Bernd Buehler, Ji Zhu, Ronda Allen. Automated walk away NGS sample preparation for constructing in-line molecular barcoded RNA libraries from Fresh Frozen and FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 764.