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Overview
Oxford Nanopore Technologies' range of sequencing kits is designed to prepare DNA and RNA libraries using 10 ng input material or more. We have now adapted a method for whole genome amplification for use with nanopore devices. The Qiagen REPLI-g Midi kit works on as little as 10 pg bacterial DNA and yields up to 40 µg DNA. After amplification, the sample is treated with T7 Endonuclease I, which resolves the hyperbranched structure of the WGA product and allows to obtain average qscores which are similar to those obtained without WGA.
Using the whole genome amplification protocol results in shorter fragment lengths - mostly up to 5 kb - than preparing a DNA library using the Ligation Sequencing Kit.
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Introduction to the whole genome amplification protocol
This protocol describes how to carry out sequencing of genomic DNA using the Ligation Sequencing Kit (SQK-LSK112). It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.
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
Library preparation
You will need to:
- Amplify the genomic DNA using random primers
- Digest the amplified DNA with T7 Endonuclease I to remove branching, and size-select for longer fragments using AMPure XP beads
- Prepare the DNA ends for adapter attachment
- 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
- Start the EPI2ME software and select a workflow for further analysis (this step is optional)