Cell free DNA (cfDNA) info sheet

Overview

Analysis of cell-free (cf)DNA can be used for a range of uses, including cancer detection and tissue-of-origin analysis.

This document is a repository for all of our internally developed methods, know-how documents and information. This will ensure our users can easily access all the required information and our recommended best practices using nanopore sequencing technology for cfDNA.

Document version: CFD_S1020_v1_revA_07Aug2024

1. Resources

End-to-end protocols

Our end-to-end protocols provide you with a step-by-step guide to carry out the preparation and sequencing of a human cfDNA sample from fresh blood or plasma.

Ligation sequencing V14 — Human cfDNA singleplex (SQK-LSK114)
Our singleplex method allows you to process a single human cell-free DNA (cfDNA) sample with our Ligation Sequencing Kit V14 (SQK-LSK114).

Typically, we obtain ~50 Gb of aligned data (15x coverage) for human cfDNA samples processed with this protocol.
Singleplex 1 output Sequencing and primary aligned output for 4x replicates of ~15 ng inputs, with each library run on a single PromethION flow cell. Where a) sequencing output averages 70 Gb, b) primary aligned data averages 52 Gb and c) read count averages 227 M.
Singleplex 2 frag size Read length profiles (Q-score ≥10) of total data output for a) Sequencing data, b) Aligned data. The marginal reduction in length of aligned data is due to the informatic removal of sequencing adapter. These profiles are equivalent for each of the 3x replicates.

Ligation sequencing V14 — Human cfDNA multiplex (SQK-NBD114.24)
Our multiplex method 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.
Multiplex 1 output Per barcode sequence data output from single PromethION flow cells, using 12x barcoded libraries (72 hour runs) (Q-score ≥10) . a) Raw sequencing data (Gb). b) Primary aligned data (Gb). 6 ng of cfDNA was required to generate >3Gb of aligned data which equates to approximately 1X human genome coverage. c) Read count (million reads).
Multiplex 2 frag size Read length profiles (Q-score ≥10) of total data output (multiplexed) for a) Sequencing data, b) Aligned data. The reduction in length of aligned data is due to the removal of native barcode and adapter sequence. These profiles are equivalent for each of the 12x demultiplexed samples.

Sample extraction methods

Our extraction methods have been developed by our internal teams to generate the best input available for your sequencing experiment. These methods use the QIAGEN QIAamp MinElute ccfDNA Midi Kit. Please note, these sample extraction methods have also been fully integrated into our end-to-end protocols.

Frag length femtopulse cfDNA singleplex extract Example of fragment length profile of extracted cfDNA using QIAGEN QIAamp MinElute ccfDNA Midi Kit, run on a Femto Pulse (Agilent). This example shows the characteristic nucleosome peaks with minimal gDNA contamination.

Know-how and information documents

Our know-how and information documents present you with additional background information on the methods we have developed and/or provide additional practices and data to optimise your sequencing experiment.

Last updated: 8/7/2024

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