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Nat Protoc. 2019 Oct;14(10):2749-2780. doi: 10.1038/s41596-019-0202-2. Epub 2019 Aug 30.

Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA.

Author information

1
Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
2
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
3
Department of Radiation Oncology, University of Toronto, Toronto, Ontario, Canada.
4
Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada. daniel.decarvalho@uhnresearch.ca.
5
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. daniel.decarvalho@uhnresearch.ca.

Abstract

Circulating cell-free DNA (cfDNA) comprises small DNA fragments derived from normal and tumor tissue that are released into the bloodstream. Recently, methylation profiling of cfDNA as a liquid biopsy tool has been gaining prominence due to the presence of tissue-specific markers in cfDNA. We have previously reported cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as a sensitive, low-input, cost-efficient and bisulfite-free approach to profiling DNA methylomes of plasma cfDNA. cfMeDIP-seq is an extension of a previously published MeDIP-seq protocol and is adapted to allow for methylome profiling of samples with low input (ranging from 1 to 10 ng) of DNA, which is enabled by the addition of 'filler DNA' before immunoprecipitation. This protocol is not limited to plasma cfDNA; it can also be applied to other samples that are naturally sheared and at low availability (e.g., urinary cfDNA and cerebrospinal fluid cfDNA), and is potentially applicable to other applications beyond cancer detection, including prenatal diagnostics, cardiology and monitoring of immune response. The protocol presented here should enable any standard molecular laboratory to generate cfMeDIP-seq libraries from plasma cfDNA in ~3-4 d.

PMID:
31471598
DOI:
10.1038/s41596-019-0202-2

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