Transcriptomic profiling of ccRCC nephrectomy samples by Direct RNAseq

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. Following primary tumour resection approximately 30% of patients experience disease recurrence associated with metastasis. To date, long-read RNA sequencing has not been applied to kidney cancer. Here, we used ONT long-read Direct RNA sequencing to profile the transcriptomes of ccRCC archival tumours, 6 of which were from patients who went on to relapse. Our results revealed a loss of immune infiltrate in tumours of patients who relapse. Moreover, thousands of novel isoforms were discovered, including a novel PD-L1 transcript encoding for the soluble version of the protein but having a longer 3'UTR than the currently annotated transcript. Finally, we have identified a novel non-coding gene that was over-expressed in patients who experience recurrence. Our data shows that DRS can be used in archival tumour samples to comprehensively characterise tumour transcriptomes, and to reveal novel features that would have been missed by short-read RNAseq. RNA was extracted from nephractomy samples, with 2 μg of total RNA per sample used as input for sequencing library preparation (RNA002). Each library was loaded on individual PromethIon flow cell (R9) and sequenced for 72h. Reads with Q >7 were used for subsequent bioinformatic analysis.