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Overview of nanopore data analysis
For a full overview of nanopore data analysis, which includes options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.
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How to start sequencing
The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software. It is assumed you have already installed MinKNOW on your computer. Instructions for this can be found in the MinKNOW protocol.
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MinKNOW settings for real-time basecalling:
We recommend setting up real-time basecalling as follows:
Real-time basecalling
Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section for your device until the end of the "Completing a MinKNOW run" section.
Select Ligation Sequencing Kit (SQK-LSK114) in "Kit selection". The barcoding option will be unavailable as default. Other parameters can be kept at their default settings.
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Post-run barcode demultiplexing:
We currently recommend performing demultiplexing for the dual barcoding protocol post-sequencing using the Guppy software.
For a complete guide please refer to our Guppy protocol
Barcode demultiplexing in Guppy for dual barcoding:
To demultiplex your reads by barcode, use the dual barcoding configuration file, specifying the barcode kit as "EXP-DUAL00":
guppy_barcoder --input_path <folder containing FASTQ and/or FASTA files> --save_path <output folder> --config configuration_dual.cfg --barcode_kits "EXP-DUAL00"
For more information and instructions on how to use Guppy, please refer to the relevant sections of the protocol: