Document Type

Article

Publication Date

2-4-2021

Keywords

JGM

JAX Source

BMC Cancer 2021 Feb 4; 21(1):114

PMID

33541297

DOI

https://doi.org/10.1186/s12885-021-07814-8

Abstract

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.

Comments

This article is licensed under a Creative Commons Attribution 4.0 International License.

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