Oxford Nanopore Sequencing Technology help

[mido1951]

Hello,
I am trying to understand the technology of sequencing Oxford Nanopore.
if 1D is sequenced from a single strand. How to build 2D from "1D Temp" and "1D Comp" ??
I speak in the image of step "G" and "H".

The explanation of the entire process is:
(A) Suspended library molecules are concentrated near nanopores embedded in the membrane. A voltage applied across the membrane induces a current through the nanopores. (B) Schematic of a library molecule, showing dsDNA ligated to a leader adapter pre-loaded with a motor protein and a hairpin adapter pre-loaded with a hairpin protein, and the tethering oligos. (C) Sequencing starts from the 5’ end of the leader adapter. The motor protein unwinds the dsDNA allowing single-stranded DNA to pass through the pore. (D) A flow cell contains 512 channels (grey), each channel consisting of 4 wells (white). Each well contains a pore (blue) and a sensor. At any given time, the device is recording the data stream from the wells of the active well-group, in this example, g1. (E) Perturbation in the current across the nanopore is measured 3,000 times per second as ssDNA passes through the nanopore. (F) The ‘bulk data’ are segmented into discrete ‘events’ of similar consecutive measurements. The 5-mer corresponding to each event is inferred using a statistical model. (G) The 1D base-calls are inferred separately for the template and complement event signals. (H) Alignment of the 2D base-calls from the event signals from both, and the 1D base-calls are used to constrain the 2D base-calls.

[gringer]

It's a consensus call of the template and complement strands (e.g. global alignment).

I don't think the method implemented by ONT pays much attention to the actual event signal, but you can certainly deal with plenty of errors by taking into account things like event length and the complementary hexamer event probability.

[mido1951]

I know there are errors.
but I do not understand step G and H.

[gringer]

Step G should really start 1 sentence earlier. The current ONT base calling procedure uses a Viterbi algorithm to find the optimal path through a Hidden-Markov model of hexamer transition probabilities.

Step H, as I've mentioned in my last post, is carried out via an alignment of the called template sequence to the called complement sequence, most likely a "quick" global alignment without accounting for base quality or signal characteristics.

If that's not helpful for a description, you'll probably need to read up on HMMs (computer science term) and global alignment (biology / genetics term). It doesn't make sense for me to reiterate other very good tutorials on the Internet.