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Important
This text and figures are based on the Oxford nanopоре documentation from the official nanoporetech site and official videos.
The basic principle of the Oxford Nanopore Technology (ONT) is straightforward. Molecules are driven through a pore in a membrane. As a molecule moves through membrane it blocks current. In fact the extent of "blockage" is sequence specific generating using current change profiles that can be "translated" into sequence.
It began as technique for measuring polynecleotide length
followed by experimentation on controlling the translocation speed using a variety of enzymes including a DNA polymerase:
ReadUntil allows "adaptive sampling" of reads in real time. In particular it makes things like UNCALLED possible:
The general idea behind UNCALLED is summarized in this figure from the above manuscript:
a, Overview of the algorithm: inputs are an FM index built from the DNA reference and the raw nanopore signal. The signal is converted to events, and the log probability of events matching each k-mer is computed. All paths through the FM index that are consistent with k-mers matching each event above a threshold are searched, conceptually forming a forest of trees (Extended Data Fig. 1 provides more details). b, Boxplots showing the speed of UNCALLED mapping of 100,000 E. coli reads to the E. coli K12 reference genome (kb s–1, left) and total number of milliseconds required to map reads (right). Center lines represent the median, box limits represent the upper and lower quartiles and whiskers represent the 5 and 95% confidence intervals. c, Percentage of mapped reads that can be confidently placed within a certain number of base pairs of sequencing. Note the ONT MinION sequences at approximately 450 bp s–1. b,c, Only reads that were mapped by UNCALLED are considered.
Open this notebook to have a look at raw data.