Robust methylation-based classification of brain tumours using nanopore sequencing.

BACKGROUND: DNA methylation-based classification of cancer provides a comprehensive molecular approach to diagnose tumours. In fact, DNA methylation profiling of human brain tumours already profoundly impacts clinical neuro-oncology. However, current implementation using hybridisation microarrays is time consuming and costly. We recently reported on shallow nanopore whole-genome sequencing for rapid and cost-effective generation of genome-wide 5-methylcytosine profiles as input to supervised classification. Here, we demonstrate that this approach allows us to discriminate a wide spectrum of primary brain tumours. RESULTS: Using public reference data of 82 distinct tumour entities, we performed nanopore genome sequencing on 382 tissue samples covering 46 brain tumour (sub)types. Using bootstrap sampling in a cohort of 55 cases, we found that a minimum set of 1000 random CpG features is sufficient for high-confidence classification by ad hoc random forests. We implemented score recalibration as a confidence measure for interpretation in a clinical context and empirically determined a platform-specific threshold in a randomly sampled discovery cohort (N = 185). Applying this cut-off to an independent validation series (n = 184) yielded 148 classifiable cases (sensitivity 80.4%) and demonstrated 100% specificity. Cross-lab validation demonstrated robustness with concordant results across four laboratories in 10/11 (90.9%) cases. In a prospective benchmarking (N = 15), the median time to results was 21.1 h. CONCLUSIONS: In conclusion, nanopore sequencing allows robust and rapid methylation-based classification across the full spectrum of brain tumours. Platform-specific confidence scores facilitate clinical implementation for which prospective evaluation is warranted and ongoing.

Fast capture and multiplexed detection of short multi-arm DNA stars in solid-state nanopores.

Fast and multiplexed detection of low-abundance disease biomarkers at the point-of-need would transform medicine. Nanopores have gained attention as single-molecule counters to electrically detect a range of biological molecules in a handheld format, but challenges remain before diagnostic applications can emerge. For solid-state nanopore sensors, the specificity of the ionic current signatures and the rate of target capture required to simultaneously recognize and rapidly count a mixture of molecular targets in a complex sample are active areas of research. Herein, we study the capture and translocation characteristics of short N-arm star shaped DNA nanostructures to evaluate their potential as a family of surrogate label molecules for biomarkers of interest, designed for fast and reliable multiplexed detection based on conductance blockages. Simple hybridization of a varying number of short, easily synthesized 50 bp ssDNA strands allows the number of arms in the star shape DNA to be controlled from N = 3 to 12. By introducing more arms to the nanostructures, we show that we can controllably increase the nanopore signal-to-noise ratio for a range of pore sizes, producing conductance blockages which increase linearly with the number of arms, and we demonstrate conductance-based multiplexing through simultaneous detection of three such nanostructures. Moreover, the increased molecular signal strength facilitates detection under salt concentration asymmetries, allowing for a capture rate enhancement of two orders of magnitude without compromising the nanopore temporal and ionic signals. Together, these attributes (strong signal, multiplexing potential and increased counting rate) make the N-arm star DNA-based nanostructures promising candidates as proxy labels for the detection of multiple biomarkers of interest in future high sensitivity single-molecule solid-state nanopore-based assays.

Capture and Translocation Characteristics of Short Branched DNA Labels in Solid-State Nanopores.

The challenge when employing solid-state nanopores as single-molecule sensors in a given assay is the specificity of the ionic current signal during the translocation of target molecules. Here we present the capture and translocation characteristics of short structurally defined DNA molecules that could serve as effective surrogate labels in biosensing applications. We produced T-shaped or Y-shaped DNA molecules with a 50 bp double-stranded DNA (dsDNA) backbone and a 25 bp dsDNA branch in the middle, as improved labels over short linear DNA fragments. We show that molecular topologies can be distinguished from linear DNA by analyzing ionic current blockades produced as these DNA labels translocate through nanopores fabricated by controlled breakdown on 10-nm-thick SiN membranes and ranging in diameter from 4 to 10 nm. Event signatures are shown to be a direct result of the structure of the label and lead to an increased signal-to-noise ratio over that of short linear dsDNA, in addition to well resolved dwell times for the pore size in this range. These results show that structurally defined branched DNA molecules can be robustly detected for a broad range of pore size, and thus represent promising candidates as surrogate labels in a variety of nanopore-based molecular or immunoassay schemes.