The nanopore sequencing workflow
Oxford Nanopore is focused on enabling the simplest possible workflows, that can be performed in any location and by people without years of wet lab skills. With that in mind, we have created 10 minute/two pot library prep, we are developing VolTRAX for automated preparation, we are continually improving the system software and providing real time analysis workflows on the EPI2ME platform.
Real time data: no fixed run time allows on-demand sequencing and real time adjustment
A key feature of the MinION device, and of the PromethION and GridION systems, is that there is no fixed run time; a user can run any of the systems for a short or long period of time, as data is streamed in real time.
This means that workflows are efficient. Users do not have to wait for sufficient samples to start a machine, as they could use only one of the five GridION flow cells, or one of the 48 PromethION flow cells. Barcoding is available, or a single sample could be applied to a flow cell, run, the flow cell washed and used again.
Real time data also enables real time insights. The user can predetermine an experimental endpoint and run the system for as long as it takes to collect sufficient data to address that question.
PromethION is designed to run up to 48 flow cells at any time, on-demand.View PromethION
During an experiment, each nanopore in the array analyses molecules in the sample independently of the other nanopores. The shortest time to start collecting experimental data is the time taken for one analyte molecule to successfully interact with one nanopore in the array. While a single DNA molecule may take milliseconds to seconds to pass through the nanopore, data starts becoming available as soon as it starts passing through the pore. When a DNA molecule has passed through a nanopore, another will load.
Data analysis takes place in real time as data streams from the sensor chip in parallel from multiple nanopores. Therefore, a longer run enables more data points to be collected, more confidence about an observation to be achieved, more measurement accuracy to be obtained, and a greater range of analyses to occur.
Run until... sufficient data
An Oxford Nanopore device may be instructed to Run until... a certain datum has been seen a certain number of times at a specified confidence level. In this way, the experiment is defined by the user, not defined by the machine.
For example: the system may process the sample until a minimum of tenfold read coverage over specified regions of interest has been seen, until a specific mutation has been observed in a sample, or until enough sequence data has been collected to reliably assemble a sample against a reference.
Real-time analyses can result in simpler workflows, for example the real-time assembly of reads during an experiment.
Read until...: real-time selective sequencing
The Read until... method enables the user to analyse only DNA strands that contain predetermined signatures of interest. The DNA sequence of the first bases of a DNA molecule to pass through a nanopore are analysed in real time. If found to be of interest, sequencing continues. If found to be not of interest to the experiment then the DNA strand can be rejected from the pore in real time, freeing that pore to sequence an alternative preferred read. This method allows selective sequencing, improving both the time to result and the efficiency of an experiment.