Application of Synthetic External RNA Controls in a Targeted Hybrid Capture Assay

Abstract

It is common practice for laboratories that run any type of diagnostic assay or molecular test at even moderate frequency to employ a well characterized in-process control sample to verify and validate assay specifications and performance. ERCC spike in mixes are a widely used set of external synthetic RNA transcripts designed to be added to in-process control RNA samples. These specific spike in mixes are most often utilized in gene expression profiling assays such as total RNA seq and microarrays to verify sensitivity, limit of detection, and reproducibility. This study investigates the feasibility of using ERCC spike in mixes to perform similar measurements in a targeted hybrid capture assay instead of whole transcriptome sequencing or microarrays. Manufacturer instructions do not provide guidance for this less common application, empirical knowledge and several rounds of testing were employed to determine optimal conditions to enable translation to a hybrid capture assay. Sensitivity, reproducibility, and sequencing read distribution were analyzed across a range of spike in mix concentrations and captured with targeted panels of varying size. Subsequent results indicate that when capturing ERCC spike in mixes with a large sized panel, the ERCC spike in mix should remain more concentrated to preserve ability to detect ERCC transcripts present at low abundance in the mix. When capturing with a smaller targeted panel, the ERCC spike in mix should be diluted to a lower concentration to avoid consuming an exorbitant amount of sequencing reads and therefore taking away valuable coverage of other targets in the panel. A linear relationship was demonstrated between spike in mix concentration, percent of sequencing reads consumed, and successful detection of ERCC transcripts present at low molecular abundance. Expression correlation between identical sample replicates demonstrated high reproducibility at every dilution of spike in mix, including the least concentrated twenty-fold dilution. The results of this study demonstrate a unique and promising application of synthetic spike in transcripts in a hybrid capture assay.