LAUSR

The RNA sequencing (RNA-Seq) technique is now routinely used to quantitatively explore genome-wide expression by various research fields including cancer research. The most common RNA-seq methodology produce billions of short-read sequencing in the range of 100–600 base pairs, from which it is occasionally difficult to reconstruct isoform-level transcriptome and fusion genes. The limitations of the short-reads can be overcome by using third-generation sequencing technologies, such as Oxford Nanopore Technologies (ONT). This study aims to perform full-length cDNA sequencing using ONT platform and investigate the abilities of ONT in (1) identifying differential gene expression, (2) detecting differential transcript isoform usage, and (3) detecting fusion genes. To do these methods, CNS-1 cells were implanted into the frontal lobes of three Lewis rats. The CNS-1 model is a histocompatible astrocytoma cell line with an invasive pattern mimicking glioblastoma (GBM). After two weeks of transplantation, the transplanted tumors and the normal brain on the other side were collected as matched normal-tumor pairs. Total RNA extracted from the samples were subjected to the full-length cDNA sequencing on a portable MinION sequencer. In tumors samples, 615 genes involved in cell cycle were upregulated, whereas 1067 genes involved in neurological functions were downregulated. Finally, we could identify differential transcript isoform expression and fusion genes from the matched normal-tumor pairs. Overall, full-length sequencing of the cDNA molecules permitted a detailed characterization of the differential gene expression, the isoform complexity, and fusion genes. In the near future, we will use these methods on human samples.