Abstract 5881: Assessment of two whole transcriptome RNA sequencing library preparation Illumina workflows in FFPE human specimens with suboptimal quality
Authors: Wang Danyi , Rolfe Alex , Huang Alice , O'Rourke Dennis , Scheuenpflug Juergen , Feng Zheng 
Published: September 2020

Abstract
Purpose High analytical sensitivity and a broad dynamic range make RNA sequencing (RNA-Seq) an appealing platform for mRNA expression analyses for clinical exploratory biomarker development and identification. However, the reliability and accuracy of RNA-Seq data rely on the input RNA template quality and quantity. Additionally, cDNA library preparation methods can significantly influence the results, especially in samples with suboptimal quality such as those originating from FFPE specimens. Here we compared two Illumina library preparation methods for RNA-Seq analysis using several FFPE samples from human cancer indications at two independent vendors.
Study Procedure Twenty-five FFPE samples from 5 indications (NSCLC, CRC, RC, BC and HCC) were used, covering a wide range of sample storage (3-25 years-old), sample qualities, and specimen types (resection vs. core needle biopsy). Each sample was processed independently by both vendors. Total RNA was isolated using the Qiagen miRNeasy FFPE kit followed by library construction using either TruSeq Stranded Total RNA library preparation kit with Ribo-Zero Gold, or TruSeq RNA Access library preparation kit. Libraries were normalized to 20pM and sequenced on an Illumina HiSeq 2500 using V3 chemistry, in paired-end mode with a read length of 2 × 50bp. The data were processed through a standard RNASeq pipeline to produce counts and transcripts per millions (TPMs) for each gene in each sample to compare two library kits at two different vendors.
Data Summary QC data for FFPE RNA extraction, library preparation and sequencing generated by the two vendors are comparable, and sequencing results show high concordance as well. With the TruSeq Total kit, the average spearman correlation between vendors was 0.87 and the average Pearson correlation was 0.76. With the Access kit, the average spearman correlation between vendors was 0.89 and the average Pearson correlation was 0.73. Illumina TruSeq RNA Access Library generates more reads from protein coding genes than the Illumina TruSeq Stranded kit and generated acceptable sequencing results at both vendors in all usable samples (15/20), based on post alignment QC mapping to a reference genome (&gt;90%).
Conclusion Our analyses provide evidence to guide selection of sequencing methods for studies in which the sample quality is severely compromised. The comparison results demonstrate that the Illumina RNA Access library protocol more efficiently targets protein coding genes than TruSeq Stranded Total RNA library protocol on highly degraded FFPE samples and can be an attractive alternative for clinical trial studies in which the FFPE quality is severely compromised. However, since the probe selection step in the RNA Access protocol may bias results compared to other platforms, further exploration may be needed.
Citation Format: Danyi Wang, Alex Rolfe, Alice Huang, Dennis O'Rourke, Juergen Scheuenpflug, Zheng Feng. Assessment of two whole transcriptome RNA sequencing library preparation Illumina workflows in FFPE human specimens with suboptimal quality [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5881.