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Unlocking the transcriptomic potential of formalin-fixed paraffin embedded clinical tissues: Comparison of gene expression profiling approaches [Lexogen_QuantSeq]

ABSTRACT: Background: High-throughput transcriptomics has matured into a very well established and widely utilised research tool over the last two decades since the first mRNA profiling microarrays. Clinical datasets generated on a range of different platforms continue to be deposited in public repositories provide an ever-growing, valuable resource for reanalysis. Cost and tissue availability normally preclude processing samples across multiple technologies, making it difficult to directly evaluate performance, reliability and to what extent gene expression data from different platforms can be compared or integrated. Purpose: In this study, we describe our experiences using nine new and established mRNA profiling techniques including Lexogen QuantSeq, Qiagen QiaSeq, BioSpyder TempO-Seq, Ion AmpliSeq, Nanostring, Affymetrix Clariom S or U133A, Illumina BeadChip and Ion Total RNA-seq of formalin-fixed paraffin embedded (FFPE) and fresh frozen (FF) sequential patient-matched breast tumour samples. Results: The number of genes represented and reliability were found to vary between the platforms, but overall all methods provided data which were largely comparable. Crucially we found that it is possible to integrate data for combined analyses across FFPE/FF and platforms using established batch correction methods as required to increase cohort sizes. However, some platforms appear to be better suited to FFPE samples, particularly archival material. Overall, we illustrate that technology selection is a balance between required resolution, sample quality, availability and cost.

ORGANISM(S): Homo sapiens

PROVIDER: GSE130469 | GEO | 2019/05/03

REPOSITORIES: GEO

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Project description:Background: The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. Results: In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. Conclusions: Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.

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Unlocking the transcriptomic potential of formalin-fixed paraffin embedded clinical tissues: Comparison of gene expression profiling approaches [Lexogen_QuantSeq]

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