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Introduction to the multiplex human cfDNA sequencing protocol
This protocol describes how to carry out preparation and sequencing of 12 human cell-free DNA (cfDNA) samples using the Native Barcoding Kit 24 V14 (SQK-NBD114.24). Typically, we obtain ~3 Gb of aligned data (1x coverage) for each of the 12 human cfDNA samples processed with this protocol.
Please note: This method has been developed for multiplexing 12 samples. We do not recommend deviating from the outlined method.
Reducing the number of samples multiplexed may lead to an increase in coverage, but increasing the number of samples multiplexed will lead to a reduction in coverage of aligned data per sample.Prior to library preparation, the sample extraction is carried out using the QIAGEN QIAamp MinElute ccfDNA Midi Kit and following our Human blood cell-free DNA (cfDNA) extraction for multiplex sequencing method.
Note: We recommend that blood samples are processed while fresh, as we have observed potential gDNA contamination arising from blood that has been stored in certain types of collection tubes.
For more information on the development and performance of this method, please refer to our Updated method for cell-free DNA (cfDNA) methylation profiling know-how document. An additional know-how document is also available for the optimisation of library preparation for longer cell-free DNA (cfDNA).
Steps in the 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
Sample preparation
Using the outlined extraction method, extract the cfDNA from your human blood samples, and quantify the DNA:
Library preparation
The table below is an overview of the steps required in the library preparation, including timings and optional stopping points.
Library preparation Process Time Stop option DNA repair and end-prep Repair the cfDNA and prepare the DNA ends for adapter attachment 125 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 Attach the sequencing adapters to the DNA ends 50 minutes 4°C short-term storage or for repeated use, such as re-loading your flow cell
-80°C for single-use, long-term storage.
We strongly recommend sequencing your library as soon as it is adapted.Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes Sequencing and analysis
You will need to:
- Start a sequencing run using the MinKNOW software which will collect raw data from the device and basecall reads.
- (Optional) Raw sequencing data can be basecalled and aligned to a reference using dorado.
- (Optional) Start the EPI2ME software and select a bioinformatics workflow to analyse your data. Alternatively, external tools can be used to further analyse and explore your data.