Published: October 31st, 2016
Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.
Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).
Epigenetic regulation of genes through DNA methylation is one of the essential mechanisms required to determine the cell fate by stable differentiation of different tissue types in the body1. Dysregulation of this process has been known to cause various diseases including cancer2.
This process mainly involves the addition of methyl groups on the cytosine residue in the CpG dinucleotides of DNA3. There are a few different techniques currently used to investigate this mechanism, each having their own advantages as outlined in many studies2-8. Here we will discuss one of these techniques called Methyl-Binding DNA Capture sequencing (MBD....
All tissues are obtained following approval of the Institutional Review Board committee and when all participants consented to both molecular analyses and follow-up studies. The protocols are approved by the Human Studies Committee at University of Texas Health Science Center at San Antonio.
1. Methyl-binding DNA Capture (MBDCap)
We have used MBDCap-seq to study DNA methylation alterations in a large number of patients from diverse cancer types including breast12, endometrial13, prostate14, and liver cancers among others. Here we demonstrate some information from the breast cancer study published recently12. In this instance, we used the whole genome sequencing approach to identify CpG islands that are differentially methylated in tumor with respect to normal across different genomic regions. The investigation revealed that.......
The MBDCap-seq technique is an affinity enrichment approach3, considered as a cost effective alternative when investigating cohorts with a large number of patients15. The pipeline presented here describes a comprehensive approach from sample procurement to data analysis and interpretation. One of the most important steps is setting up a PCR amplification procedure to improve the PCR efficiency of the GC enriched regions in the genome as this is where DNA methylation occurs. Also, it is essential to ensure that after seq.......
The work is supported by CPRIT Research Training Award RP140105, as well as partially supported by US National Institutes of Health (NIH) grants R01 GM114142 and by William & Ella Owens Medical Research Foundation.....
|Methylminer DNA enrichment Kit
|Dynabeads M-280 Streptavidin
|Bioruptor Plus Sonication Device
|3M sodium acetate pH 5.2
|SPRIworks Fragment Library System I
|Fully automated library construction system
|PCR primer mix
|Fluorometric Quantitation System
|PCR master mix
|PCR master mix
|PCR Purification beads
|HiSeq 2000 Sequencing System
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