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Introduction to the protocol
This protocol describes an end-to-end workflow for telomere-to-telomere sequencing of the human genome using the Oxford Nanopore PromethION platform. The protocol includes three separate sequencing experiments, each requiring at least one PromethION Flow Cell, which are carried out over seven days. The library preparation, sequencing and flow cell washing and reloading steps are described day-by-day.
This protocol was developed in collaboration with the UCSC Nanopore Production Center, led by Dr. Karen Miga.
To generate as much data as possible, multiple flow cell washes are required to recover pores and reload fresh library to maximise data output. Depending on sample quality and DNA yield post-library preparation, the library preparation steps may need to be scaled up to meet the number of fresh libraries required for each flow cell wash and library reload. Excess DNA libraries can be stored in Eppendorf DNA LoBind tubes at 4°C until loading on the required day.
Three different datasets are generated to give both high accuracy duplex data and ultra-long reads, alongside the chromatin conformation capture data to achieve in-depth telomere-to-telomere sequencing of a sample.
Three experiments are set up across seven days:
Duplex experiment:
This experiment generates high accuracy duplex data using the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL). Library preparation takes ~60 minutes hands-on time, followed by three washes across a 100-hour sequencing run.Ultra-long DNA experiment:
This experiment generates ultra-long reads using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114). DNA extraction and library preparation takes ~3.5 hours with an overnight elution, followed by two washes across a 72-hour sequencing run.
This experiment generates a very viscous library of very long DNA fragments which requires careful handling to maintain the long fragments.Pore-C experiment:
This experiment produces chromatin conformation capture data using the Pore-C protocol and the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL). The Pore-C DNA extraction takes ~3 hours hands-on time over three days with two overnight steps. The library preparation takes ~60 minutes hands-on time, followed by three washes across a 72-hour sequencing run.
This experiment has been developed by Oxford Nanopore Technologies and the following published literature: Lieberman-Aiden et al., 2009; Comet et al., 2011; Belton et al., 2012; Gavrilov, Golov and Razin, 2013; Nagano et al., 2015; Belaghzal, Dekker and Gibcus, 2017; Ulahannan et al., 2019. This experiment intends to manipulate cell suspensions extracted from whole blood to capture three-dimensional interactions of DNA within chromatin. This workflow has been written using NlaIII restriction enzyme and the heat denaturation method. For further information on protocol considerations, please see the Restriction Enzyme Pore-C info sheet.
Steps in the sequencing workflow
Prepare for your experiment
You will need to:- Prepare your samples for all experiments.
- Ensure you have your sequencing kits, the correct equipment and third-party reagents.
- Download the software for acquiring and analysing your data.
- Check your flow cells to ensure they have enough pores for a good sequencing run.
Library preparation
Duplex experiment:
You will need to:- Repair the DNA, and prepare the DNA ends for adapter attachment
- Attach sequencing adapters to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cell
- Wash and reload the flow cell approximately every 20-24 hours
Ultra-long DNA experiment:
You will need to:- Extract your uHMW gDNA
- Tagment your DNA using a diluted fragmentation mix
- Attach the sequencing adapters to the DNA ends
- Clean-up the sample by precipitating your DNA and elute overnight
- Prime the flow cell and load your DNA library into the flow cell
- Wash and reload the flow cell approximately every 20-24 hours
Pore-C experiment:
You will need to:- Crosslink the three dimensional DNA interactions within the nucleus
- Permeabilise the cells to expose the crosslinked structures and denature the chromatin
- Cleave the genome into clusters of crosslinked DNA fragments at recognition sites with a restriction enzyme
- Ligate the cohesive ends of proximal crosslinked monomers into chimeric Pore-C polymers held in proximity
- Degrade the protein structures to release the chimeric Pore-C polymers into solution
- Purify the Pore-C extract before DNA repair and end-prep for adapter attachment
- Attach sequencing adapters to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cell
- Wash and reload the flow cell approximately every 18 hours
Sequencing and analysis
You will need to:
- For each experiment, start a sequencing run using the MinKNOW software which will collect raw data from the device and convert it into basecalled reads.
- For each experiment, we recommend basecalling in real-time during sequencing before rebasecalling and aligning to a reference genome post-sequencing. Further details for each experiment set up are outlined in the "Sequencing and data analysis" section.