Real‐time analysis of the cancer genome and fragmentome from plasma and urine cell‐free DNA using nanopore sequencing

Abstract

Cell‐free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short‐read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor‐derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated ( R = 0.98) with the tumor fraction calculated from short‐read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment‐end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor‐derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost‐effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations. image Cell‐free DNA (cfDNA), a rising biomarker in oncology, can be used as a readout from a liquid biopsy. Current analytical methods employ short‐read DNA sequencing technologies. We designed a long‐read nanopore sequencing technique to analyze liquid biopsy samples. Tumor‐derived cfDNA could be detected in the plasma and urine of cancer patients using nanopore sequencing, demonstrating sensitivity equivalent to short‐read sequencing. The turnaround time from sample collection to obtaining results was < 24 h when utilizing the deployable MinION Nanopore platform. Nanopore sequencing successfully captured fragmentomics signal, including long cfDNA fragments (> 300 bp), in both plasma and urine samples. The long cfDNA fragments (ranging from > 300 to 8,055 bp) contained tumor‐derived molecules, as confirmed in human and xenograft samples.