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Introduction to the adeno-associated virus sequencing protocol
This end-to-end protocol describes how to extract recombinant adeno-associated virus (rAAV) vectors using the PureLink™ Viral RNA/DNA Mini extraction kit before sequencing using the Native Barcoding Sequencing Kit 24 V14 (SQK-NBD114.24). We have also included an optional annealing step post-extraction, however, we have found higher amounts of full-length inverted terminal repeat (ITR) sequences when the annealing step has been skipped before library preparation. Flushing steps have also been included as we recommend washing the flow cell to restore pores and to load a fresh library to continue sequencing.
The Know-How document is available for further details about the protocol optimisations and best practices.
Note: This protocol is currently validated to barcode up to six AAV samples for sequencing on a single flow cell.
Sequencing of the rAAV vectors enables the validation of vectors to ensure the transgene and promoter of interest are present, as well as identifying truncated rAAV genomes and any contamination. Validation is crucial in gene therapy to ensure the correct rAAV genomes are packaged into cells before therapeutic use.
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
Protocol step Process Time Stop option DNAseI treatment Perform DNAseI treatment of the rAAV lysates to remove any non-encapsidated DNA from the rAAV preparations 35 minutes - DNA extraction from rAAV Extract the rAAV vectors using the PureLink™ Viral RNA/DNA Mini Kit 45 minutes –80°C for long-term storage Annealing (Optional) Self-anneal any remaining (+) and (-) single strands of rAAV vector 80 minutes - End-prep Prepare the DNA ends for adapter attachment 20 minutes 4°C overnight Native barcode ligation Ligate the native barcodes to the DNA ends 60 minutes 4°C overnight Adapter ligation and clean-up Ligate sequencing adapters to the DNA ends 50 minutes 4°C for short-term storage or for repeated use, such as for reloading your flow cell
–80°C for long-term storagePriming and loading the flow cell Prime the flow cell, and load your DNA library into the flow cell 5 minutes Sequencing
You will need to:
- Start a sequencing run using the MinKNOW software which will collect raw data from the device and basecall reads in real-time. The reads will also be demultiplexed in MinKNOW.
- Start the EPI2ME software and use the wf-aav-qc workflow for analysis.