Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples

Yulia Newton, Andrew J. Sedgewick, Luis Cisneros, Justin Golovato, Mark Johnson, Christopher W. Szeto, Shahrooz Rabizadeh, J. Zachary Sanborn, Stephen Charles Benz, Charles Vaske

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

Original languageEnglish (US)
Article number17597
JournalScientific reports
Volume10
Issue number1
DOIs
StatePublished - Dec 1 2020
Externally publishedYes

ASJC Scopus subject areas

  • General

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