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Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients [CRC]

ABSTRACT: Genome-wide analysis of cell-free DNA (cfDNA) methylation profile has been recognized as a promising approach for sensitive and specific detection of many cancers. However, scaling such genome-wide assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We have shown that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation with high sensitivity and quantitative tracking of colorectal cancer patient’s response to treatment, at much reduced sequencing cost suitable for frequent monitoring. We also show, for the first time, tumor epigenetic subtyping from cfDNA using Heatrich-BS, which could enable patient stratification from non-invasive liquid biopsy. As such, Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE208596 | GEO | 2022/07/21

REPOSITORIES: GEO

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Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients

Project description:Genome-wide analysis of cell-free DNA (cfDNA) methylation profile has been recognized as a promising approach for sensitive and specific detection of many cancers. However, scaling such genome-wide assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We have shown that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation with high sensitivity and quantitative tracking of colorectal cancer patient’s response to treatment, at much reduced sequencing cost suitable for frequent monitoring. We also show, for the first time, tumor epigenetic subtyping from cfDNA using Heatrich-BS, which could enable patient stratification from non-invasive liquid biopsy. As such, Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

2022-05-13 | GSE202606 | GEO

Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients

Project description:Genome-wide analysis of cell-free DNA (cfDNA) methylation profile has been recognized as a promising approach for sensitive and specific detection of many cancers. However, scaling such genome-wide assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We have shown that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation with high sensitivity and quantitative tracking of colorectal cancer patientÃ¢Â€Â™s response to treatment, at much reduced sequencing cost suitable for frequent monitoring. We also show, for the first time, tumor epigenetic subtyping from cfDNA using Heatrich-BS, which could enable patient stratification from non-invasive liquid biopsy. As such, Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

| EGAS00001006198 | EGA

Single-stranded Pre-methylated 5mC Adapters Uncover the Methylation Profile of Plasma Ultrashort Single-stranded Cell-free DNA

Project description:Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ~50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the “5mCAdpBS-Seq” workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (~15%) and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment positively affected gene expression of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

2024-03-28 | GSE252088 | GEO

Genome-wide plasma cell-free DNA methylation profiling in lung cancer patients using MeDIP-seq

Project description:As a non-invasive blood testing, the detection of cell-free DNA (cfDNA) methylation in plasma is raising increasing interest due to its diagnostic and biology applications. Although extensively used in cfDNA methylation analysis, bisulfite sequencing is less cost-effective. Through enriching methylated cfDNA fragments with MeDIP followed by deep sequencing, we aimed to characterize cfDNA methylome in cancer patients. In this study, we investigated the cfDNA methylation patterns in lung cancer patients by MeDIP-seq. MEDIPS package was used for the identification of differentially methylated regions (DMRs) between patients and normal ones. Overall, we identified 330 differentially methylated regions (DMRs) in gene promoter regions, 33 hypermethylation and 297 hypomethylation respectively, by comparing lung cancer patients and healthy individuals as controls. The 33 hypermethylation regions represent 32 genes. Some of the genes had been previously reported to be associated with lung cancers, such as GAS7, AQP10, HLF, CHRNA9 and HOPX. Taken together, our study provided an alternative method of cfDNA methylation analysis in lung cancer patients with potential clinical applications.

2019-03-31 | E-MTAB-7163 | biostudies-arrayexpress

Bisulfite amplicon sequencing can detect Glia and Neuron cell-free DNA in blood plasma.

Project description:Sampling the live brain is difficult and dangerous, and withdrawing cerebrospinal fluid is uncomfortable and frightening to the subject, so new sources of real-time analysis are constantly sought. Cell-free DNA (cfDNA) derived from glia and neurons offers the potential for wide-ranging neurological disease diagnosis and monitoring. However, new laboratory and bioinformatic strategies are needed. DNA methylation patterns on individual cfDNA fragments can be used to ascribe their cell-of-origin. Here we describe bisulfite sequencing assays and bioinformatic processing methods to identify cfDNA derived from glia and neurons. In proof-of-concept experiments we describe the presence of both glia- and neuron-cfDNA in the blood plasma of human subjects following mild trauma. These detection of glia- and neuron-cfDNA represents a significant step forward in the translation of liquid biopsies for neurological diseases.

2021-02-06 | GSE126663 | GEO

Clinical Study of Pan-cancer DNA Methylation Test in Plasma

Project description:We intend to establish an efficient method for plasma cfDNA extraction and Bisulfite transformation to facilitate the detection of DNA methylation status using multiplex fluorescence PCR. Meanwhile, we expect to identify several plasma methylation markers that can be highly sensitive for multi-cancer detection. Finally, we will provide a pan-cancer blood test that is easy to operate, low cost, accurate and easy to promote.

| 5068 | ecrin-mdr-crc

CUT&Tag-BS: an efficient and low-cost method for simultaneous profiling of histone modification and DNA methylation

Project description:It remains a challenge to decipher the complex relationship between DNA methylation, histone modification, and the underlying DNA sequence with limited input material. Here, we developed an efficient, low-input, and low-cost method for simultaneous profiling of genomic binding sites of histone modification and methylation status of the underlying DNA at single-base resolution from the same cells in a single experiment by integrating CUT&Tag with tagmentation-based bisulfite sequencing (CUT&Tag-BS). We demonstrated the validity of our method for both active and repressive histone modifications using 250,000 mouse ESCs. CUT&Tag-BS showed similar enrichment patterns of histone modification to those observed in non-bisulfite-treated control; it further revealed that H3K4me1-marked regions are mostly CpG-poor, lack of methylation concordance, and exhibit prevalent DNA methylation heterogeneity among the cells. We anticipate that CUT&Tag-BS will be widely applied to directly address the genomic relationship between DNA methylation and histone modification, especially in low-input scenario with precious biological samples.

2021-10-20 | GSE179266 | GEO

A comprehensive DNA methylation atlas for noncancer human tissue types

Project description:Deciphering the tissue origin of cfDNA can reveal abnormal cell death because of diseases, which has great clinical potential in disease detection and monitoring. Here we present one of the largest comprehensive and high-resolution methylation atlas based on Reduced Representative Bisulfite Sequencing (RRBS) data of 521 noncancer tissue samples spanning 29 major types of human tissues. We systematically identified fragment-level tissue-specific methylation patterns and extensively alidated the methylation signature atlas in independent methylation datasets, orthogonal epigenomic markers, and transcription regulatory elements. Based on the rich tissue methylation atlas, we develop the first supervised tissue deconvolution approach, a deep-learning-powered model, cfSort, for sensitive and accurate tissue deconvolution in cfDNA.

2023-06-15 | GSE233417 | GEO

Cell-free DNA comprises an in vivo, genome-wide nucleosome footprint that informs its tissue(s)-of-origin

Project description:Nucleosomes are the basic unit of packaging of eukaryotic chromatin, and nucleosome positioning can differ substantially between cell types. Here, we sequence 14.5 billion plasma-borne cell-free DNA (cfDNA) fragments (700-fold coverage) to generate genome-wide maps of in vivo nucleosome occupancy. We identify 13 million local maxima of nucleosome protection, spanning 2.53 gigabases (Gb) of the human genome, whose positions and spacings correlate with nuclear architecture, gene structure and gene expression. We further show that short cfDNA fragments - poorly recovered by standard protocols - directly footprint the in vivo occupancy of DNA-bound transcription factors such as CTCF. The sequence composition of cfDNA has previously been used to noninvasively monitor cancer, pregnancy and organ transplantation, but a key limitation of this paradigm is its dependence on genotypic differences to distinguish between contributing tissues. We show that nucleosome spacing in gene bodies and cis-regulatory elements, inferred from cfDNA in healthy individuals, correlates most strongly with transcriptional and epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as the normal source of cfDNA. We build on this observation to show how in vivo nucleosome footprints can be used to infer the cell types that contribute to circulating cfDNA in pathological states such as cancer. Because it does not rely on genotypic differences, this strategy may enable the noninvasive cfDNA-based monitoring of a much broader set of clinical conditions than is currently possible. Sequencing of cfDNA libraries from healthy individuals, pooled healthy individuals and individuals with disease for the identification of nucleosomes and protection from other DNA binding proteins.

2015-11-04 | E-GEOD-71378 | biostudies-arrayexpress

Discovery of Methylated Circulating DNA Biomarkers For Comprehensive Non-Invasive Monitoring Of Treatment Response In Metastatic Colorectal Cancer.

Project description:Evaluation of cell free circulating DNA (cfDNA) has recently been proposed for tracking disease relapse in colorectal cancer (CRC). Such assays could overcome the issues of classical monitoring, but requires personalized assay design due to absence of common alteration. On the contrary, early methylation alterations are restricted to delimited genomic loci allowing comprehensive assay design for population study. Our objective was to identify new cancer specific methylation biomarkers fitted to assess cfDNA over treatment, thus monitoring response in the metastatic CRC (mCRC) setup. Genome wide methylation profiling of colorectal cancer cell lines (surrogate to pure tumor tissue) was carried out and compared to publically available normal healthy colon mucosa (cancer unrelated). Identified loci were validated in additional publically available cohorts. Digital PCR assays were designed for selected markers and assessed in cfDNA of self-declared healthy donors (>40 years old), and of mCRC patients. Longitudinal assessment was performed in a subset of colorectal cancer patients treated with different therapy regimen (chemotherapy or targeted agents). Samples from the current GEOset were extracted by Wizard SV Genomic DNA Purification System (Promega). DNA samples were checked for integrity by 1% agarose gel, and quantity via Picogreen quantification. All samples were bisulfite converted using the EZ DNA methylation Gold kit (Zymo Research) with a minimum input of 500ng and following manufacturer’s instructions. Infinium HumanMethylation450 BeadChip arrays were carried out according to the Illumina Infinium HD methylation protocol.

2017-10-10 | GSE86078 | GEO

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