Nano-RAPID: Integrated Diagnosis for Sarcoma Using a Nanopore Sequencer

Pathological diagnosis of sarcoma is challenging. Although high-throughput sequencing and methylation array aid sarcoma diagnosis and are routine in high-income countries, access to these genetic/epigenetic analyses in many countries, including Japan, remains limited. Nanopore sequencing is an easy, fast, and cost-effective high-throughput sequencing alternative. Nanopore sequencers read long-read DNA to detect fusion DNA and 5-methyl-cytosine in single nucleotide resolution, enabling real-time basecalling for selective enrichment of regions of interest via "adaptive sampling." Nanopore sequencing of Raw DNA with Adaptive sampling for Prompt Integrated Diagnosis (Nano-RAPID) was developed to test its diagnostic utility. This study included 22 retrospective and nine prospective cases. DNA extracted from fresh/frozen tissue was sequenced for 46-98 h, followed by approximately 12 h of data analysis. Consequently, disease-specific/characteristic fusion genes were detected in 10 retrospective and six prospective cases. Disease-specific single nucleotide variants were detected in three cases, while disease-specific amplifications were detected in two retrospective cases. Monosomy, trisomy, and chromosomal instability were also detected. Methylation analyses accurately classified 15 retrospective and five prospective cases. Collectively, correct tumor types were identified using at least one modality in 19 retrospective and nine prospective cases. Halving the consumables and re-analyzing 24-h data yielded nearly equivalent results, suggesting faster and cost-effective analyses. In conclusion, Nano-RAPID may be a promising adjunctive for sarcoma diagnosis. Nano-RAPID could be introduced in low-equipped hospitals to assist non-specialized pathologists diagnosing sarcoma. Since this study remains exploratory, further studies with larger cohort should be needed.