Methyl-binding DNA capture Sequencing for Patient Tissues

Summary

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.

Abstract

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).

Introduction

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 studies^2-8. Here we will discuss one of these techniques called Methyl-Binding DNA Capture sequencing (MBD....

Protocol

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)

1. Sample collection and DNA isolation
   1. Collect bulk tumor or normal tissue samples from patient paraffin embedded tissue samples.
   2. Use a commercial DNA mini kit to isolate genomic DNA from the paraffin embedded tissue samples according to manufacturer's protocol.
   3. Use a meth....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Methylminer DNA enrichment Kit	Invitrogen	ME10025
Dynabeads M-280 Streptavidin	Invitrogen	112-05D
Bioruptor Plus Sonication Device	diagenode	B01020001
3 M sodium acetate, pH 5.2	Sigma	S7899	100 mL
SPRIworks Fragment Library System I	Beckman Coulter	A50100	Fully automated library construction system
Adapter Primers	Bioo Scientific	514104	PCR primer mix
Qubit	Invitrogen	Q32854	Fluorometric Quantitation System
PCR master mix	KAPA scientific	KK2621	PCR master mix
AMPure XP	Beckman Coulter	A63881	PCR Purification beads
EB Buffer	Qiagen	19086
HiSeq 2000 Sequencing System	Illumina