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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
The table below is an overview of the steps required in the sample and library preparation, including timings and optional stopping points.
Library preparation Process Time Stop option For gDNA: DNA end-prep
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For amplicons: Incorporate tailed primersPrepare the DNA ends for PCR adapter attachment
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Perform a round of PCR to incoporate tailed primers35 minutes 4°C overnight For gDNA: attach barcoding adapters
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For amplicons: second round of PCR to incorporate barcode sequencesAttach barcoding adapters to the DNA ends.
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Complete a second round of PCR to incorporate the Oxford Nanopore barcode sequences.40 minutes 4°C overnight Amplify and pool samples Amplify each barcoded sample by PCR, then pool the samples together. 15 minutes + Adjustable PCR time 4°C overnight End-prep Repair the DNA and prepare the DNA ends for adapter attachment 35 minutes 4°C overnight Adapter ligation and clean-up Attach the sequencing adapters to the DNA ends 20 minutes 4°C short-term storage or for repeated use, such as re-loading your flow cell
-80°C for single-use, long-term storage.
We strongly recommend sequencing your library as soon as it is adapted.Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes 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).