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Ultra-Long DNA Sequencing Kit features:
This kit is recommended for users who:
- Want to reliably generate ultra-long read length N50s (>50 kb), with yields of 50-100+ Gbases on PromethION
- Sequence long reads from extracted uHMW DNA
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Introduction to the Ultra-Long DNA Sequencing Kit protocol (SQK-ULK114)
This protocol describes the complete workflow from extracting gDNA from frozen tissue or purified cells from whole blood to the sequencing of ultra-high molecular weight (uHMW) gDNA using the Ultra-Long DNA Sequencing Kit (SQK-ULK114). We have also included the procedure to isolate white blood mononuclear (WBC) from whole blood and how to quantify gDNA developed by Paul A ‘Giron’ Koetsier & Eric J Cantor, 2021.
We have used the NEB Monarch® HMW DNA Extraction Kit for Tissue (cat # T3060) to extract the uHMW gDNA for both input types when developing this protocol. Alternative kits are available from NEB which are specifically designed for the extraction from blood and cells. However, they have not been validated by Oxford Nanopore Technologies.
Per reaction, there is enough library generated for multiple consecutive loads onto a flow cell to increase output. To load a library three times on a PromethION flow cell, a flow cell wash is required to recover channels.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:- If working with whole blood, isolate white blood cells. If working with frozen tissue, isolate cells from the tissue
- Extract your uHMW gDNA
- Quantify your sample
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- If not already installed, download the software for acquiring and analysing your data
- Check your flow cell(s) to ensure it has enough pores for a good sequencing run
Library preparation
You will need to:- Tagment your DNA using a diluted fragmentation mix
- Attach Rapid Adapters to the DNA ends
- Clean-up your sample by precipitating your DNA and elute overnight
- 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
- Optional: Start the EPI2ME software and select a workflow for further analysis
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Flow cell loading and flushing
The Ultra-Long DNA Sequencing Kit (SQK-ULK114) protocol generates viscous DNA which can affect flow cell loading. We have modified the flow cell loading steps to take account for this. Please take care and follow the steps carefully to avoid damaging the flow cell.
To increase output, we recommend loading an ultra-long library three times per flow cell. A flow cell wash using the Flow Cell Wash Kit (EXP-WSH004) is required between each subsequent library load to recover channels. To run a second library straight away, please follow the modified method in this protocol: To run another library of ultra-long DNA on a PromethION flow cell straight away.
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Best practice for handling uHMW gDNA
When mixing, we recommend using wide-bore pipette tips to mix the full volume of a sample to ensure thorough mixing whilst minimising mechanical shearing of long fragments.
To preserve longer DNA, mix slower and more gently. Vortexing on low speeds may also be used at the expense of very long fragments.
While precautions should be taken to ensure that DNA fragment lengths are preserved, there should be no compromise to ensuring that reagents are thoroughly mixed with DNA. Insufficient mixing will lead to reduced read length and output.
For further information, please refer to the troubleshooting section.