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Introduction to the Four-primer PCR protocol
This protocol describes the step-by-step instructions for amplicon sequencing using the PCR Sequencing Kit (SQK-PSK004) or the PCR Barcoding Kit (SQK-PBK004), and the Four-primer PCR method.
- Please take note, from Batch 12 (SW04.10.0012) onwards of the PCR Barcoding Kit (SQK-PBK004), 1D Barcode Primer 01-12 (LWB 01-12) has changed name to Barcode Primer 01-12 (BP 01-12).
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 runLibrary preparation
You will need to:
- Perform a four-primer PCR reaction using your own specific primers, and primers supplied in the kit
- Attach sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cellFigure 1. Four-primer PCR to generate specific targets with 5’ tags for simplified post-PCR sequencing adapter attachment.
a) Users tail the 5’ end of their own primers with a specific sequence. These “inner” primers amplify the target of choice. Oxford Nanopore supplies a set of “outer” primers which prime off the tail on the 5’ end of the inner primers. Thus in a single PCR, the target amplicon with the required 5’ tag, is generated.
b) To enable multiplexing of the PCR products on the same flow cell, targets can be amplified using barcoded “outer” primers. The protocol has been developed with a starting quantity of 30 ng DNA. However, you are welcome to optimise the protocol for other starting amounts of DNA; adjust the number of PCR cycles accordingly.Sequencing and analysis
You will need to:
- Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
- optional Start the EPI2ME software and select a workflow for further analysis, e.g. metagenomic analysis or drug resistance mapping