Roche Sequencing Solutions, Inc.

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Morgan Mager

John Mannion

John S Foster

Improved multi-cell nanopore-based sequencing chips and methods can employ formation, characterization, calibration, and/or normalization techniques. For example, various methods may include one or more steps of performing physical checks of cell circuitry, forming and characterizing a lipid layer on the cells, performing a zero point calibration of the cells, forming and characterizing nanopores on the lipid layers of each cell, performing a sequencing operation to accumulate sequencing signals from the cells, normalizing those sequencing signals, and determining bases based on the normalized sequencing signals.

Formation and calibration of nanopore sequencing cells

Techniques for measuring sequences of nucleic acids are provided. Time-based measurements (e.g., forming a histogram) particular to a given sequencing cell can be used to generate a tailored model. The model can include probability functions, each corresponding to different states (e.g., different states of a nanopore). Such probability functions can be fit to a histogram of measurements obtained for that cell. The probability functions can be updated over a sequencing run of the nucleic acid so that drifts in physical properties of the sequencing cell can be compensated. A hidden Markov model can use such probability functions as emission probabilities for determining the most likely nucleotide states over time. For sequencing cells involving a polymerase, a 2-state classification between bound and unbound states of the polymerase can be performed. The bound regions can be further analyzed by a second classifier to distinguish between states corresponding to different bound nucleotides.

Basecalling for stochastic sequencing processes

A nanopore cell includes a conductive layer and a working electrode disposed above the conductive layer and at the bottom of a well into which an electrolyte may be contained, such that at least a portion of a top base surface area of the working electrode is exposed to the electrolyte. The nanopore cell further includes a first insulating wall disposed above the working electrode and surrounding a lower section of a well, and a second insulating wall disposed above the first insulating wall and surrounding an upper section of the well, forming an overhang above the lower section of the well. The upper section of the well includes an opening that a membrane may span across, and wherein a base surface area of the opening is smaller than the at least a portion of the top base surface area of the working electrode that is exposed to the electrolyte.

Small aperture large electrode cell

A nanopore cell is disclosed. The nanopore cell includes an electrolyte well having a bottom base, a surrounding sidewall, and a hydrophobic surface above the surrounding sidewall. The nanopore cell further includes a first layer of electrode material disposed on the bottom base of the electrolyte well. The nanopore cell further includes a second layer of electrode material disposed on the surrounding sidewall of the electrolyte well and electrically connected to the first layer of electrode material. The first layer of electrode material and the second layer of electrode material are configured to jointly provide capacitive coupling when an electrolyte is placed in the electrolyte well.

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Sunnyvale, CA, United States of America

Morgan Mager