BMC Cancer 2021 Feb 4; 21(1):114
BACKGROUND: Our objective was to assess whether modifications to a customized targeted RNA sequencing (RNAseq) assay to include unique molecular identifiers (UMIs) that collapse read counts to their source mRNA counts would improve quantification of transcripts from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. The assay (SET4) includes signatures that measure hormone receptor and PI3-kinase related transcriptional activity (SET
METHODS: Modifications included steps to introduce eight nucleotides-long UMIs during reverse transcription (RT) in bulk solution, followed by polymerase chain reaction (PCR) of labeled cDNA in droplets, with optimization of the polymerase enzyme and reaction conditions. We used Lin's concordance correlation coefficient (CCC) to measure concordance, including precision (Rho) and accuracy (Bias), and nonparametric tests (Wilcoxon, Levene's) to compare the modified (NEW) SET4 assay to the original (OLD) SET4 assay and to whole transcriptome RNAseq using RNA from matched fresh frozen (FF) and FFPE samples from 12 primary breast cancers.
RESULTS: The modified (NEW) SET4 assay measured single transcripts (p< 0.001) and SET
CONCLUSIONS: Modifications to the targeted RNAseq protocol for SET4 assay significantly increased the precision of UMI-based and reads-based measurements of individual transcripts, multi-gene signatures, and mutant transcript fraction, particularly with FFPE samples.
Fu, Chunxiao; Marczyk, Michal; Samuels, Michael; Trevarton, Alexander J; Qu, Jiaxin; Lau, Rosanna; Du, Lili; Pappas, Todd; Sinn, Bruno V; Gould, Rebekah E; Pusztai, Lajos; Hatzis, Christos; and Symmans, W Fraser, "Targeted RNAseq assay incorporating unique molecular identifiers for improved quantification of gene expression signatures and transcribed mutation fraction in fixed tumor samples." (2021). Faculty Research 2021. 58.