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PCR Barcoding Expansion features:
We have two PCR barcoding expansions available depending on the number of barcodes required:
- PCR Barcoding Expansion 1-12 (EXP-PBC001): up to 12 unique barcodes are available
- PCR Barcoding Expansion 1-96 (EXP-PBC096): up to 96 unique barcodes are available
These expansions are used with the Ligation Sequencing Kit V14 and recommended for users who:- Want to multiplex up to 12 or 96 barcodes, depending on the expansion they are using.
- Would like to achieve raw read sequencing modal accuracy of Q20+ (99%) or above.
- Want to optimise their sequencing experiment for output.
- 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 PCR Barcoding protocol
This protocol describes how to carry out PCR barcoding of gDNA using the Ligation Sequencing Kit V14 (SQK-LSK114) with the PCR Barcoding Expansion Pack 1-12 (EXP-PBC001) or 1-96 (EXP-PBC096). This protocol also outlines recommendations to PCR barcode amplicons. Using the PCR Barcoding expansions allows for up to either 12 or 96 samples to be combined and loaded onto a single flow cell. It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.
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
Experiment workflow
Prepare your gDNA input:- Prepare the DNA ends for adapter attachment
- Attach barcoding adapters to the DNA ends
- Amplify each barcoded sample by PCR, then pool the samples together
OR prepare your amplicon input:- Perform a round of PCR to incorporate tailed primers
- Complete a second round of PCR to incorporate the Oxford Nanopore barcode sequences and amplify each barcoded sample, then pool the samples together
Library preparation:- Prepare the DNA ends for adapter attachment
- Attach sequencing adapters to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cell
Sequencing and analysis
You will need to:
- In the current MinKNOW software version, the PCR Barcoding Expansions are not available in "Kit selection" when setting up a sequencing run. These will be included in the next software update. For the meantime, we recommend the following:
- Start a sequencing run in the MinKNOW software using SQK-LSK114, which will collect raw data from the device and convert it into basecalled reads.
- Demultiplex your data post-run on MinKNOW using the barcoding option, choosing either EXP-PBC001 or EXP-PBC096 as your barcoding kit. Further information is available in the "Post-run barcoding" of the MinKNOW protocol.
- Start the EPI2ME software and select the barcoding workflow for further analysis (this step is optional).