Transcriptomic profiling of ccRCC nephrectomy samples by PCR-cDNAseq

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 PCR-cDNAseq 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 ONT long-read PCR-cDNAseq 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 (PCS111). Each library was loaded on individual PromethIon flow cell (R9) and sequenced for 72h. Reads with Q >7 were used for subsequent bioinformatic analysis.