Fig. 1

Flow-chart of library optimization and bioinformatics evaluation. a A pilot study consisting of FFPE and fresh frozen pairs for 7 BBD patients were submitted for sequencing to evaluate two protocols of library preparation for RNA-seq, Ribo-depletion and RNA exome capture. Several bioinformatics metrics were evaluated for the two protocols. Whole exome sequencing (WES) data was used to estimate SNP confirmation rate, and the RNA exome capture showed superior performance in all categories and was selected as the library preparation protocol to process all samples. b 130 study samples (ER+ estrogen receptor positive, ER− estrogen receptor negative, Cont control) along with 17 technical replicates and 11 study replicates were submitted for library preparation using the RNA exome capture protocol. 40 samples failed library preparation step with insufficient RNA. All remaining samples were submitted for sequencing in 10 batches. Rigorous bioinformatics evaluation was performed to identify qc failed samples based on defined bioinformatics metrics. The final dataset comprised 62 study samples