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Introduction to amplicon sequencing using the Rapid Barcoding Kit 24 or 96 V14
This protocol describes how to carry out rapid barcoding of amplicon DNA using the Rapid Barcoding Kit 24 or 96 V14 (SQK-RBK114.24 or SQK-RBK114.96) to sequence up to 96 single amplicon samples. This method allows you to perform your own amplicon sequencing and validation with a quick turn-around time, without the need for primers and a reference, and with competitive price per sample. Using this method, PCR amplicons ranging from 500 bp to 5 kb can be sequenced, which allows users to check that each amplicon is the correct size and no mutations have been introduced during PCR amplification.
The analysis workflow is not intended for marker gene sequencing of mixtures/communities of different organisms (e.g. 16S sequencing). In de-novo consensus mode it expects a single amplicon per barcode. When running in variant calling mode, multiple amplicons per barcode can be processed, but their sequences need to be sufficiently different from each other so that most reads only align to one of the provided references.
We recommend new users to sequence for 12 hours, although a shorter run-time (e.g. 4 hours) may be sufficient to generate enough reads per target. We suggest generating 150X or ~1500 reads per target. In most cases, this should be sufficient data to perform analysis.
After sequencing, we recommend performing downstream analysis using the EPI2ME amplicon workflow (wf-amplicon).
The results of the workflow include an interactive HTML report, FASTQ files with the consensus sequences of the amplicons, and BAM files with alignments of the input reads re-aligned against the consensus. Optionally, a reference with the expected amplicon sequences can be supplied to the workflow, in which case VCF files with variants called against that reference are additionally emitted.
Detailed instructions for setting up MinKNOW and the EPI2ME workflow are included in this protocol.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your DNA, and check its length, quantity and purity using the Input DNA/RNA QC protocol. The quality checks performed during the protocol are essential in ensuring experimental success.
- Ensure your primer design is correct prior to amplicon generation.
Note: To make sure that the whole region of interest is captured when using Rapid Barcoding Kits, the primers should be designed such that they include an extra of 15-20 bp at the start and end of the actual target sequence. - Generate your amplicon sample(s) by PCR amplification.
- 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
The table below is an overview of the steps required in the library preparation, including timings and stopping points.
Library preparation step Process Time Stop option PCR clean-up AMPure XP Bead purification (or equivalent) of amplicon samples to remove PCR artifacts 25 minutes 4°C overnight Amplicon DNA barcoding Tagmentation of the amplicon DNA using the Rapid Barcoding Kit V14 15 minutes 4°C overnight Sample pooling and clean-up Pooling of barcoded libraries and AMPure XP Bead clean-up 25 minutes 4°C overnight Adapter ligation Attach the sequencing adapters to the DNA ends 5 minutes 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:
- Start a sequencing run using the MinKNOW software, which will collect raw data from the device into basecalled reads and will perform barcode demultiplexing.
- Start the EPI2ME software and use the EPI2ME amplicon workflow (wf-amplicon) for analysis.