Real-time analysis of the cancer genome and fragmentome from plasma and urine short and long cell-free DNA using Nanopore sequencing

Abstract

Cell-free DNA (cfDNA) can be isolated from blood and/or urine of cancer patients and analyzed with sequencing. Unfortunately, most conventional short-read sequencing methods are technically challenging, labor intensive and time consuming, requiring several days but more typically weeks to obtain interpretable data which are limited by a bias for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies sequencing we can achieve economical and ultra-fast delivery of clinical data from liquid biopsies. Our ITSFASTR approach is able to deliver copy number aberrations, and cfDNA fragmentation profiles in less than 24 hours from sample collection. The tumor-derived cfDNA fraction calculated from lung cancer patient plasma and urine from bladder cancer patients was highly correlated (R=0.98) to the tumor fraction calculated from conventional short-read sequencing of the same samples. cfDNA size profile and fragmentation patterns in plasma and urine exhibited the typical cfDNA features yet with a significantly higher proportion of fragments that exceed 300bp, exhibiting similar tumor fraction than shorter size fragments. Notably, comprehensive fragment-end composition and nucleosome profiling near transcription start sites can be determined from the same data. We propose that ITSFASTR is the first point-of-care solution for obtaining genomic and fragmentomic results from liquid biopsies.

Long-read sequencing reveals aberrant fragmentation patterns and origins of circulating DNA in cancer

“…Longer fragments, including those greater than 1kb, were under-represented. Similar patterns have been described previously both with Illumina sequencing [14,15] and ONT sequencing [25,26]. Interestingly, PC3-high cancers tended to have lower frequencies of the DNASE1L3-associated CC end motifs (Figure 4C).…”

“…While several studies have used ONT and Pacific Biosciences sequencing to investigate biological properties of longer fragments >500bp [24,[26][27][28][29], the issue of genomic contamination has not been addressed. Previous work has shown that genomic contamination occurs as longer fragments and can depend on pre-analytical processing, especially in cryo-preserved samples [32,33].…”

“…While fragmentomic features of ctDNA are of great interest, much of our understanding comes from Illumina short-read sequencing. Recently, amplification-free sequencing of longer circulating DNA fragments in cancer has been demonstrated using Oxford Nanopore Technologies (ONT) and Pacific Biosciences sequencing [23][24][25][26][27][28]. While these technologies broadly recapitulate fragmentomic and other features associated with cancer [25], they also provide an opportunity to expand our understanding and reexamine earlier findings.…”

Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling

“…Moreover, cost-effectiveness is only achieved if samples are processed in large batches, resulting in prolonged turn-around times of several weeks, which is often incompatible with clinical timelines. In contrast, Oxford Nanopore Technologies (ONT) Nanopore sequencing offers low-cost, portable, and non-batched sequencing and is therefore increasingly utilized for cfDNA sequencing (van der Pol et al 2023; Lau et al 2023; Marcozzi et al 2021). Copy number alterations and fragmentomic features can be determined confidently in liquid biopsies with a ctDNA fraction of ∼3% and higher using nanopore sequencing (van der Pol et al 2023).…”

“…In contrast, Oxford Nanopore Technologies (ONT) Nanopore sequencing offers low-cost, portable, and non-batched sequencing and is therefore increasingly utilized for cfDNA sequencing (van der Pol et al 2023; Lau et al 2023; Marcozzi et al 2021). Copy number alterations and fragmentomic features can be determined confidently in liquid biopsies with a ctDNA fraction of ∼3% and higher using nanopore sequencing (van der Pol et al 2023). The main challenge for implementing nanopore sequencing for the purpose of ctDNA sequencing is the relatively high single-nucleotide error rate (Marcozzi et al 2021).…”