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Features of this protocol:
This protocol is recommended for users who want to incorporate unique molecular identifiers (UMIs) into targeted amplicons.
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Introduction to the protocol
This protocol may be used to incorporate unique molecular identifiers (UMIs) into amplicons to be sequenced using the Ligation Sequencing Kit (SQK-LSK109). Custom primers are used to target a specific locus for amplification and tag amplicons with UMIs. Subsequent rounds of universal amplification synthesise UMI amplicon copies which may then be sequenced. The resulting 1D reads are then clustered together based on their UMI identity to generate a single high accuracy read for each original UMI tagged molecule.
This protocol has been developed based on research by Oxford Nanopore Technologies and published literature: Søren M. Karst, Ryan M. Ziels, Rasmus H. Kirkegaard, Emil A. Sørensen, Daniel McDonald, Qiyun Zhu, Rob Knight and Mads Albertsen (2020) “Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing”. bioRxiv, 645903. doi: https://doi.org/10.1101/645903.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Design and order primers as further explained in Primer Design of this document
- Extract your DNA and check its length, quantity and purity. The quality checks are 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
Library preparationYou will need to:
- Fragment and UMI tag the DNA
- Cycle 1: The top and bottom target strands are unilaterally tagged with a UMI tailed with a universal priming site
- Cycle 2: The opposite terminus is tagged to complete a dsDNA template bilaterally tagged with complimentary UMI sequences tailed with universal priming sites
- Amplify the DNA and prepare the DNA ends for adapter attachment
- The intermediate AMPure XP bead cleans between the early and late PCR steps reduce the presence of off-target amplification
- Attach sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell and load your DNA library into the flow cell
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