A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

2019 ◽  
Author(s):  
A. De Koker ◽  
R. Van Paemel ◽  
B. De Wilde ◽  
K. De Preter ◽  
N. Callewaert
Keyword(s):  
Bisulfite Sequencing ◽  
Preparation Method ◽  
Cost Effective ◽  
Cell Free Dna ◽  
Reduced Representation ◽  
Free Dna ◽  
Genome Wide ◽  
Reduced Representation Bisulfite Sequencing ◽  
Methylation Profiling ◽  
Circulating Cell Free Dna

AbstractThe methylation profile of circulating cell-free DNA (cfDNA) in blood can be exploited to detect and diagnose cancer and other tissue pathologies and is therefore of great diagnostic interest. There is an urgent need for a cost-effective genome-wide methylation profiling method that is simple, robust and automatable and that works on highly fragmented cfDNA. We report on a novel sample preparation method for reduced representation bisulfite sequencing (RRBS), rigorously designed and customized for minute amounts of highly fragmented DNA. Our method works in particular on cfDNA from blood plasma. It is a performant and cost-effective methodology (termed cf-RRBS) which enables clinical cfDNA epigenomics studies.

scholarly journals Heatrich-BS enables efficient CpG enrichment and highly scalable cell-free DNA methylation profiling

2021 ◽  
Author(s):  
Elsie Cheruba ◽  
Ramya Viswanathan ◽  
Pui Mun Wong ◽  
Howard John Womersley ◽  
Yiting Lau ◽  
...  
Keyword(s):  
Bisulfite Sequencing ◽  
Tumor Burden ◽  
Response To Treatment ◽  
Cell Free Dna ◽  
Patient Response ◽  
Free Dna ◽  
Genome Wide ◽  
Quantitative Monitoring ◽  
Genome Bisulfite Sequencing ◽  
Methylation Profiling

Genome wide analysis of cell-free DNA (cfDNA) methylation profile has been shown to be a promising approach for sensitive and specific multi-cancer detection. However, scaling these assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We showed that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbored 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 enables focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable high accuracy and sensitivity in tumor burden estimation and quantitative monitoring of colorectal patient response to treatment, at much reduced sequencing requirement. Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

scholarly journals Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA: a proof-of-principle study

2019 ◽  
Author(s):  
Ruben Van Paemel ◽  
Andries De Koker ◽  
Charlotte Vandeputte ◽  
Lieke van Zogchel ◽  
Tim Lammens ◽  
...  
Keyword(s):  
Minimally Invasive ◽  
Bisulfite Sequencing ◽  
Methylation Pattern ◽  
Histopathological Diagnosis ◽  
Cell Free Dna ◽  
Reduced Representation ◽  
Pediatric Solid Tumors ◽  
Free Dna ◽  
Reduced Representation Bisulfite Sequencing

AbstractIn the clinical management of pediatric solid tumors, histological examination of tumor tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumor is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner.Translational relevanceObtaining a correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. Furthermore, the diagnostic odyssey can result in anxiety and discomfort for these children. By applying a novel technique, reduced representation bisulfite sequencing on cell-free DNA (cf-RRBS), we show the feasibility of obtaining the histopathological diagnosis with a minimally invasive test on either plasma or cerebrospinal fluid. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Given that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in difficult cases.

scholarly journals Alterations of 5-hydroxymethylation in circulating cell-free DNA reflect molecular distinctions of subtypes of non-Hodgkin lymphoma

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Brian C.-H. Chiu ◽  
Chang Chen ◽  
Qiancheng You ◽  
Rudyard Chiu ◽  
Girish Venkataraman ◽  
...  
Keyword(s):  
B Cell Lymphoma ◽  
Cell Free Dna ◽  
Invasive Approach ◽  
Non Invasive ◽  
Signature Genes ◽  
Non Hodgkin Lymphoma ◽  
Free Dna ◽  
Genome Wide ◽  
Circulating Cell Free Dna

AbstractThe 5-methylcytosines (5mC) have been implicated in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the role of 5-hydroxymethylcytosines (5hmC) that are generated from 5mC through active demethylation, in lymphomagenesis is unknown. We profiled genome-wide 5hmC in circulating cell-free DNA (cfDNA) from 73 newly diagnosed patients with DLBCL and FL. We identified 294 differentially modified genes between DLBCL and FL. The differential 5hmC in the DLBCL/FL-differentiating genes co-localized with enhancer marks H3K4me1 and H3K27ac. A four-gene panel (CNN2, HMG20B, ACRBP, IZUMO1) robustly represented the overall 5hmC modification pattern that distinguished FL from DLBCL with an area under curve of 88.5% in the testing set. The median 5hmC modification levels in signature genes showed potential for separating patients for risk of all-cause mortality. This study provides evidence that genome-wide 5hmC profiles in cfDNA differ between DLBCL and FL and could be exploited as a non-invasive approach.

Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

2011 ◽  
Vol 6 (4) ◽  
pp. 468-481 ◽  
Author(s):  
Hongcang Gu ◽  
Zachary D Smith ◽  
Christoph Bock ◽  
Patrick Boyle ◽  
Andreas Gnirke ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Reduced Representation ◽  
Reduced Representation Bisulfite Sequencing ◽  
Dna Methylation Profiling ◽  
Genome Scale ◽  
Methylation Profiling
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