Abstract 5781: Development of RNA-Seq library preparation method for highly degraded FFPE RNA and ultra-low RNA input
Authors: Star Ekaterina , Karim Noreen , DeGroen Berlin , Salgado Michael , Wang David , Li Jing , Ilic Katica , Beaudoin Sarah , Vakulskas Christopher , Min Bosun , Chen Peilin 
Published: March 2024

Abstract
RNA sequencing (RNA-Seq) has been widely used for investigating genetic variations, splicing, and gene expression in cancer research. However, RNA-Seq library preparation from archived formalin fixed paraffin embedded (FFPE) samples tends to fail due to both limited quantity and high degradation. We aimed to overcome the sample quality and quantity limitations by leveraging both in-house cDNA synthesis modules and the unique, single-stranded ligation strategy of the IDT xGen cfDNA & FFPE DNA Library Prep chemistry and workflow. This approach delivers high library conversion of input molecules and maximizes high quality data from sequencing. To represent the types of challenging cancer samples that may be lost during traditional RNA-Seq analyses, we collected thirty-seven low quality RNA FFPE samples (RIN &lt;3) from lung, breast, colorectal, thyroid tumors and more. In addition, twenty-one frozen tissue RNA samples and Nine RNA reference materials were used as controls. Regardless of sample origin, 25 ng of total RNA were taken for RNA-Seq library preparation. In-house cDNA synthesis modules were used for the 1st and 2nd strand cDNA synthesis followed by Illumina library generation using IDT’s xGen™ cfDNA & FFPE DNA Library Prep Kit. All sample types generated sufficient high-quality libraries (&gt;1 ug) for hybridization capture enrichment. The RNA-seq libraries were captured using IDT’s custom designed panel with 56 known fusion targets. We detected all expected fusion targets from the tested samples. No correlation was observed between sample quality and the number of genes detected between the different types of samples including the low quality FFPE. To test the lower limits of the workflow, a degraded FFPE sample and a Universal Human Reference RNA (UHR) were used to construct libraries with inputs from 1ng to 10pg. High quality libraries were successfully constructed with as low as 10pg of UHR and 50pg of the FFPE RNA sample. We demonstrated the flexibility of our current cfDNA & FFPE DNA Library Prep whose high conversion rate could be used for constructing libraries from low quality and degraded FFPE RNA in conjunction with our in-house cDNA synthesis modules. Libraries were successfully constructed from ultralow RNA input which could be leveraged for cfRNA applications. The high RT efficiency enables processing FFPE samples with very low-quality scores and provides users with an effective solution for their degraded RNA samples challenges.
Citation Format: Ekaterina Star, Noreen Karim, Berlin DeGroen, Michael Salgado, David Wang, Jing Li, Katica Ilic, Sarah Beaudoin, Christopher Vakulskas, Bosun Min, Peilin Chen. Development of RNA-Seq library preparation method for highly degraded FFPE RNA and ultra-low RNA input [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5781.