A robust targeted sequencing assay with simple, automated set up that provides accurate and sensitive measurement of gene expression from FFPE has been developed, referred to as NPSeq™. This assay is based on HTG's nuclease protection array-based qNPA™ assay, which has been launched as a diagnostic and translational research platform automated on the HTG EDGE System. Briefly, FFPE is lysed and processed through the standard qNPA protocol on EDGE, then unique barcodes, adaptors and primer seqeuences are added for sequencing. Samples are pooled, concentrated, and run on a gel for QC and clean-up before submitting for colony formation and sequencing. NPSeq retains S1 hydrolysis for specificity, while eliminating the array and hybridization capture/detection specificity steps from the qNPA protocol, replacing them with direct sequencing and counting of each nuclease protection probe. NPSeq measurement from FFPE is accurate, sensitive, and quantitatively reproducible. Performance has been evaluated using a focused assay measuring gene expression. Accuracy, defined as the correlation of measurements from frozen sample to matched FFPE, is R2 > 0.95. Compared to the qNPA ArrayPlate diagnostic assay, measuring clinical FFPE and using in each case 0.25 cm2 area of a 5 micron thick breast cancer section, the correlation of measurements was R2 = 0.93. The NPSeq assay conserves sample, with high sensitivity to routinely use small amounts of FFPE (less than 0.25 cm2 area of a 5 micron section). NPSeq was quantitative and highly reproducibility, with avg CV's of 3%. NPSeq is a true universal assay because it can measure sets of a few genes to sets of 1,000 or more genes without any change in protocol or platform. Thus, it can be used for biomarker discovery and then those biomarkers can be focused into a drug discovery or diagnostic assay without any change in reagents, protocol or platform, while delivering the same quantitative and ultra-high quality data. Since NPSeq samples can be tagged and pooled before sequencing the sequencing cost/sample can be reduced substantially, making both biomarker discovery and targeted diagnostic sequencing assays cost efficient. NPSeq exploits the qNPA advantage of a lysis only, extraction-free protocol to simultaneously measure mRNA and miRNA dependably from FFPE even where the mRNA is degraded. Additional application results from retrospective studies of clinical FFPE will be presented that validate the performance of the NPSeq assays, including an NPSeq assay containing content of the miRBase version 19 of ∼2000 miRNA and mRNA housekeeper genes that is in development and being used for miRNA biomarker identification from FFPE.
Citation Format: Debrah Thompson, Ihab Botros, Bruce Seligmann. Targeted gene expression sequencing from FFPE: NPSeq™. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4146. doi:10.1158/1538-7445.AM2013-4146