Introduction to nanopore sensing
The GridION™ system
- GridION™ informatics
- Monitoring, controlling and programming nodes
- Real-time bioinformatics
- Data handling
- DNA sequencing informatics
The MinION™ device: a miniaturised sensing system
Analytes and Applications: DNA, RNA, proteins
Fields of use
Base-calling and determination of quality metrics for sequencing experiments are performed in connection with primary data analysis, during which raw electrical signals are processed and reduced to events indicative of the four DNA or four RNA nucleobases. The results of this primary analysis are output in FASTQ file format.
Oxford Nanopore works with the Accelrys Pipeline Pilot® NGS Collection software, which already uses some of the most widely accepted alignment and assembly components and allows new ones to be added. Pre-configured Pipeline Pilot data analysis workflows running on the Pipeline Pilot server will automatically load the FASTQ data into a designated sequence alignment or assembly component selected from the Accelrys Pipeline Pilot NGS Collection or Oxford Nanopore’s own collection of algorithms (the ONT Collection). Results are output in standard BAM/SAM format.
Alignment or assembly – secondary data analysis – proceeds in line with primary data analysis as the experiment run continues. Results, which on GridION nodes are available immediately after production of the underlying raw data, can be monitored and evaluated in real time for experimental conditions and/or performance requirements according to the experiment’s designated 'Run Until….' criteria. These criteria will generally be pre-configured within the experiment’s Pipeline Pilot workflow script so that the appropriate response is triggered automatically.
Examples of Run Until …. Criteria and Corresponding Programmed Responses
|Mean Depth greater than X||Stop experiment once sufficient mean depth over the whole genome or a representative region reaches a predefined value|
|Depth over all genes is greater than X||Stop experiment once all genes are covered at a depth of at least X|
|Quality Control: if mean quality score for a certain percentage of bases is < QX||Discontinue running experiment|
|Consensus accuracy: Measure consensus accuracy by combining Q scores and coverage||Stop runs only when acceptance criteria for consensus accuracy have been reached|
|X number of bases with Q score > Y||Stop run when criteria satisfie|
The Pipeline Pilot server hosts a data repository that can include reference sequence data and other public or private data, as well as the user’s GridION system data. These data sources can all be used in evaluating data from an in-progress GridION system run against Run Until… criteria, or during post-run data analysis.
If no Run Until… criteria are designated for the experiment, the user may choose to store the aligned or assembled data in the Pipeline Pilot repository where it can be accessed at a later time for further analysis.