-
How to start sequencing ultra-long read data
The sequencing device control, data acquisition and real-time simplex basecalling are carried out by the MinKNOW software.
Ensure you are using the most recent software version of MinKNOW.
We recommend basecalling with the high accuracy (HAC) basecaller in real-time using the PromethION 24 or 48 device. After basecalling, the simplex data can be rebasecalled using the super accurate (SUP) model and aligned to a reference genome.
For detailed instructions for setting up the device and sequencing run, see the MinKNOW protocol section for "Starting a sequencing run with PromethION 24 and 48".
-
MinKNOW settings for the Ultra-long DNA experiment
For the Ultra-long DNA experiment, we recommend keeping the sequencing parameters to their default settings for simplex basecalling.
Below are the current recommendations:
Poisitions
Flow cell position: [User defined]
Experiment name: [User defined]
Flow cell type: FLO-PRO114M
Sample ID: [User defined]
Kit selection:
Kit selection: Ultra-Long DNA Sequencing Kit V14 (SQK-LSK114)
Sequencing speed: Default (400 bps)
Run options:
Run duration: 72 hours
Minimum read length: 1000 bp
Adaptive sampling
Enrich or deplete sequencing: Off
Barcoding balance: Not applicableAdvanced options
Active channel selection: On
Time between pore scans: 1.5 hours
Reserve pores: OnAnalysis:
Basecalling
Basecalling: On
Modified bases: On with '5mC' selected
Model: High-accuracy basecalling (HAC)Barcoding
Barcoding: Disabled
Alignment
Reference sequence: Off
Note: We do not currently recommend live alignment during sequencing as it can slow down system processing.Output:
Output format
Raw reads: On
.FAST5: On
.POD5: Off
.FASTQ: On
.BAM: OnFiltering: On
Qscore: 9
Read length: Unfiltered
Read splitting: Enabled
Override read splitting min score: On