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Dual Barcoding V14 features:
This protocol requires the use of two kits to generate the dual barcoded libraries:
- PCR Barcoding Expansion 1-96 (EXP-PBC096): up to 96 unique barcodes are available. These PCR barcodes are used as the primary "inner" barcodes.
- Native Barcoding Kit 24 V14 (SQK-NBD114.24): up to 24 unique barcodes are available. These native barcodes are used as the secondary "outer" barcodes.
These kits are used in successive order and recommended for users who:- Want to multiplex up to 2,304 samples, depending on the barcodes they are using from each expansion.
- Would like to achieve raw read sequencing modal accuracy of Q20+ (99%) or above.
- Require control over read length.
- Would like to utilise upstream processes such as size selection or whole genome amplification.
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Introduction to the V14 dual barcoding protocol
This protocol allows massively parallel sequencing of up to 2,304 samples (gDNA or amplicons) on a single flow cell. It uses both the PCR Barcoding Expansion 1-96 (EXP-PBC096) and the Native Barcoding Kit 24 V14 (SQK-NBD114.24).
Samples are initially PCR barcoded with the PCR Barcoding Expansion 1-96 (EXP-PBC096), allowing up to 96 samples to be pooled together. There can be up to 24 pools of 96 samples. Each pool of 96 samples will then undergo secondary barcoding through the ligation of one of 24 native barcodes using the Native Barcoding Kit 24 V14 (SQK-NBD114.24). After secondary barcoding, all pools are combined into a single library for sequencing.
Note: For amplicon inputs, first-round PCR product with the following tailed primers are required. Please see the equipment and consumables page for further information.
5’ TTTCTGTTGGTGCTGATATTGC-[ project-specific forward primer sequence ] 3’
5’ ACTTGCCTGTCGCTCTATCTTC-[ project-specific reverse primer sequence ] 3’Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your DNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- Download the software for acquiring and analysing your data
- Check your flow cell to ensure it has enough pores for a good sequencing run
Library preparation
You will need to:
- Prepare the DNA ends for adapter attachment
- Attach barcoding adapters supplied in the 96 PCR Barcoding kit to the DNA ends
- Amplify each barcoded sample by PCR, then pool the 96 samples together (up to 24 pools of 96 samples can be generated)
- Prepare the DNA ends of your pooled samples for native barcode attachment
- Ligate native barcodes to the DNA ends for each pool of 96 samples
- Pool up to 24 native barcoded libraries together
- Attach sequencing adapters supplied in the kit to the DNA ends of your combined dual barcoded libraries
- Prime the flow cell, and load your dual barcoded DNA library into the flow cell
Sequencing and analysis
You will need to:
- In the current MinKNOW software version, we recommend setting up live basecalling during the sequencing run without live barcoding. Demultiplexing of the dual barcoded reads will be carried out post-run:
- Start a sequencing run in the MinKNOW software using SQK-LSK114, which will collect raw data from the device and basecall the reads.
- Use the Guppy software to demultiplex the barcoded reads.
- Start the EPI2ME software and select the barcoding workflow for further analysis (this step is optional).