Point-of-Care Testing : One View of Past, Present, and Future Challenges

The Oxford Nanopore MinION sequences individual DNA molecules using an array of pores that read nucleotide identities based on ionic current steps. We evaluated and optimized MinION performance using M13 genomic dsDNA. Using expectation-maximization (EM) we obtained robust maximum likelihood (ML) estimates for read insertion, deletion and substitution error rates (4.9%, 7.8%, and 5.1% respectively). We found that 99% of high-quality ‘2D’ MinION reads mapped to reference at a mean identity of 85%. We present a MinION-tailored tool for single nucleotide variant (SNV) detection that uses ML parameter estimates and marginalization over many possible read alignments to achieve precision and recall of up to 99%. By pairing our high-confidence alignment strategy with long MinION reads, we resolved the copy number for a cancer/testis gene family (CT47) within an unresolved region of human chromosome Xq24.

Microbiologic cultures, the current gold standard diagnostic method for invasive Candida infections, have low specificity and take up to 2-5 days to grow. We present the results of the first extensive multicenter clinical trial of a new nanodiagnostic approach, T2 magnetic resonance (T2MR), for diagnosis of candidemia.

Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length that can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides the foundation for nanopore sequencing of long, complex, natural DNA strands.