Nanopore Sequencing of the phi X 174 genome

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. All methods and data are made fully available. Main Text: DNA sequencing is revolutionizing biomedical and other life sciences research through its expanding scope (1) and has a rapidly growing presence in clinical medicine (2). These developments are driven in part by the successful completion of the Human Genome Project (3) and in part by the introduction of new sequencing technologies that have dramatically reduced the cost of DNA sequencing (4). Although such ‘next-generation’ sequencing technologies have matured considerably since early proof-of-concepts (5-7), nearly all remain limited to short sequence reads (with the exception of real-time sequencing from elongating polymerases (8)) and rely on complex, expensive instrumentation. Most platforms are also limited with respect to speed and require extensive sample preparation steps prior to sequencing. Nanopore sequencing, independently proposed by Church and Deamer in the mid-1990s, has tremendous potential to overcome these limitations and achieve long reads, low cost, and high speed while requiring minimal sample preparation and instrumentation (i.e. ‘tricorder’-like DNA sequencing devices) (9-12). However, this promise has faced substantial technical challenges, such that despite nearly 20 years of effort, nanopore-derived sequence reads that align to complex, natural DNA sequences have yet to be demonstrated. In nanopore devices directed at DNA sequencing, a salt solution is divided into cis and trans wells by a thin membrane. A single nanometer-scale pore in the membrane connects the cis and trans wells electrically. When a voltage is applied across this membrane, ion current flows through the pore; this current provides the primary signal. DNA is negatively charged and is electrophoretically attracted into the pore. When single-stranded (ss) DNA enters the pore, it blocks some fraction of the ion current. The fraction of the ion current that is blocked depends on the identity of nucleotides within the pore (13-15). Key challenges of this technique are single-