Limitations of Detecting Genetic Variants from the RNA-Seq Data in Tissue and FNA Samples

Dr. Cihan Kaya, Ms. Princesca Dorsaint, Ms. Stephanie Mercurio, Mr. Alexander M Campbell, Mr. Kenneth Wha Eng, Dr. Marina N. Nikiforova, Dr. Olivier Elemento, Dr. Yuri E. Nikiforov, and Dr. Andrea Sboner


Genetic profiling of resected tumor or biopsy samples is increasingly used for cancer diagnosis and selecting therapy for thyroid and other cancer types. Although mutations occur in cell DNA and are typically detected using DNA sequencing, recent attempts focused on detecting pathogenic variants from RNA. The aim of this study was to determine the completeness of capturing mutations using RNA sequencing in thyroid tissue and fineneedle aspiration (FNA) samples


To compare the detection rate of mutations between DNA and RNA sequencing, 35 tissue samples were analyzed in parallel by whole exome DNA sequencing (WES) and whole transcriptome RNA sequencing (RNA-Seq) at two study sites. Then, DNA and RNA from 44 thyroid FNA and 47 tissue samples were studied using both targeted DNA sequencing and RNA-Seq.


Out of 162 genetic variants identified by WES of DNA in 35 tissue samples, 77 (48%) were captured by RNA-Seq, with a detection rate of 49% at site #1 and 46% at site #2 and no difference between thyroid and non-thyroid samples. Targeted DNA sequencing of 91 thyroid tissue and FNA samples detected 118 pathogenic variants, of which 57 (48%) were identified by RNA-Seq. For DNA variants present at >10% AF, the detection rate of RNASeq was 62% and for those at low (5-10%) AF the detection rate of RNASeq was 7% (p10% AF and 11% of mutations present at 5-10% AF were captured by RNA-Seq. As expected, none of TERT promoter mutations were identified by RNA-Seq. The rate of mutation detection by RNA-Seq was lower in FNA samples than in tissue samples (32% vs 49%, p=0.02).


In this study, RNA-Seq analysis detected only 46-49% of pathogenic variants identifiable by sequencing of tumor DNA. Detection of mutations by RNASeq was more successful for mutations present at a high allele frequency. Mutations were more often missed by RNA-Seq when present at low frequency or when tested on FNA samples. All TERT mutations were missed by RNA-Seq. These data suggest that RNA-Seq does not detect a significant proportion of clinically relevant mutations and should be used with caution in clinical practice for detecting DNA mutations.

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