Abstract
We have developed a novel nuclease protection targeted RNA sequencing assay (NPSeq™). We used NPSeq to measure miRNA (miRBase 19) levels in formalin fixed paraffin embedded (FFPE) brain tissue from deceased Alzheimer's patients and normal subjects to identify biomarkers and demonstrate feasibility. In addition to miRNA, the Ver19 NPSeq assay utilizes mRNA housekeepers for normalization purposes. NPSeq uses an extraction free lysis process followed by a nuclease protection assay. Synthetic DNA nuclease protection probes are hybridized to target RNA in the sample, then subjected to nuclease digestion. The surviving probes undergo PCR to add adapters and generate the sequencing library. Assay performance was evaluated. Accuracy making measurements from matched frozen and FFPE tissue produced R2 values >0.95. Average CVs of 3% reflected the reproducibility between samples. NPSeq is highly sensitive, allowing utilization of small amounts of FFPE, (i.e. 0.1 cm2 of a 5 micron thick section), without losing measurement of low expressed RNAs, and without any pre-amplification of the RNA. Cross-platform comparison to qPCR using reference prostate and liver RNA resulted in an R2 of 0.75 (all data) or 0.9 (outlier removed). Multiple samples were barcoded and pooled into a single sequencing run, lowering the usual sequencing cost/sample. Data analysis was easily accomplished. This is a targeted assay in which the synthetic nuclease protection probes form the sequencing library, and thus all sequences, are known. FASTQ files from the sequencer are simply compared to a look-up table of the probes, using the Bowtie short read aligner, and exact matches are counted. Metrics of the NPSeq assay and results demonstrating its use to identify miRNA biomarkers from archived FFPE brain tissue of Alzheimer's patients will be presented. The assay is also being used to identify cancer biomarkers and for the measurement of focused sets of mRNA and miRNA from clinical samples.