Comparative analysis of Illumina and Ultima-Genomics sequencing for plasma cell-free small RNA profiling in pancreatic cancer

Plasma-derived cell-free small non-coding RNAs are promising non-invasive biomarkers for cancer detection and monitoring. However, variability in sequencing output limits standardization, and cross-platform performance for plasma small RNA profiling has not been systematically evaluated. Illumina short-read sequencing is the current standard, whereas the newcomer, Ultima-Genomics platform, has been less extensively studied for circulating small RNA. To directly compare platform performance, we sequenced plasma cell-free RNA from 39 patients with pancreatic cancer and 39 healthy controls on both platforms. Ultima-Genomics generated approximately five-fold more reads, whereas Illumina achieved slightly higher enrichment efficiency and mapping rates, and the two platforms differed in isomiR end-variant calling. Depth-matched analysis showed that the broader detection was almost entirely attributable to sequencing depth rather than platform chemistry. Mean microRNA expression correlated strongly between platforms, although per-sample agreement was moderate and poor for low- and medium-abundance microRNAs. Differential expression identified 15 significant microRNAs on both platforms with concordant directions of change, most with prior records in pancreatic cancer databases. These findings indicate that both Illumina and Ultima-Genomics are suitable for plasma small RNA profiling and independently recover the same disease-associated signal, but that portability between platforms is analysis-dependent and should not be assumed uniformly across analysis types.