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 &#38; Ella Owens Medical Research Foundation.

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)
<ol>
	<li>Sample collection and DNA isolation
	<ol>
		<li>Collect bulk tumor or normal tissue samples from patient paraffin embedded tissue samples.</li>
		<li>Use a co...

<ol>
	<li>Trimarchi, M. P., Mouangsavanh, M., Huang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+epigenetics:+a+perspective+on+the+role+of+DNA+methylation+in+acquired+endocrine.">Cancer epigenetics: a perspective on the role of DNA methylation in acquired endocrine.</a> Chin. J. Cancer. 30, 749-756 (2011).</li><li>Nair, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+methyl-DNA+immunoprecipitation+(MeDIP)+and+methyl-CpG+binding+domain+(MBD)+protein+capture+for+genome-wide+DNA+methylation+analysis+reveal+CpG+sequence+coverage+bias.">Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias.</a> Epigenetics. 6, 34-44 (2011).</li><li>Zuo, T., Tycko, B., Liu, T. M., Lin, J. J., Huang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+DNA+methylation+profiling.">Methods in DNA methylation profiling.</a> Epigenomics. 1, 331-345 (2009).</li><li>Clark, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+whole+genome+approach+of+MeDIP-seq+to+the+targeted+approach+of+the+Infinium+HumanMethylation450+BeadChip((R))+for+methylome+profiling.">A comparison of the whole genome approach of MeDIP-seq to the targeted approach of the Infinium HumanMethylation450 BeadChip((R)) for methylome profiling.</a> PLoS One. 7, e50233 (2012).</li><li>Walker, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+profiling:+comparison+of+genome-wide+sequencing+methods+and+the+Infinium+Human+Methylation+450+Bead+Chip.">DNA methylation profiling: comparison of genome-wide sequencing methods and the Infinium Human Methylation 450 Bead Chip.</a> Epigenomics. , 1-16 (2015).</li><li>Huang, Y. W., Huang, T. H., Wang, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+DNA+methylomes+from+microarray+to+genome-scale+sequencing.">Profiling DNA methylomes from microarray to genome-scale sequencing.</a> Technol. Cancer Res. Treat. 9, 139-147 (2010).</li><li>Serre, D., Lee, B. H., Ting, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MBD-isolated+Genome+Sequencing+provides+a+high-throughput+and+comprehensive+survey+of+DNA+methylation+in+the+human+genome.">MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome.</a> Nucleic Acids Res. 38, 391-399 (2010).</li><li>Brinkman, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+DNA+methylation+profiling+using+MethylCap-seq.">Whole-genome DNA methylation profiling using MethylCap-seq.</a> Methods. 52, 232-236 (2010).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LOcating+non-unique+matched+tags+(LONUT)+to+improve+the+detection+of+the+enriched+regions+for+ChIP-seq+data.">LOcating non-unique matched tags (LONUT) to improve the detection of the enriched regions for ChIP-seq data.</a> PLoS One. 8, e67788 (2013).</li><li>Gu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CMS:+a+web-based+system+for+visualization+and+analysis+of+genome-wide+methylation+data+of+human+cancers.">CMS: a web-based system for visualization and analysis of genome-wide methylation data of human cancers.</a> PLoS One. 8, e60980 (2013).</li><li>Lan, X., Bonneville, R., Apostolos, J., Wu, W., Jin, V. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=W-ChIPeaks:+a+comprehensive+web+application+tool+for+processing+ChIP-chip+and+ChIP-seq+data.">W-ChIPeaks: a comprehensive web application tool for processing ChIP-chip and ChIP-seq data.</a> Bioinformatics. 27, 428-430 (2011).</li><li>Jadhav, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+DNA+methylation+analysis+reveals+estrogen-mediated+epigenetic+repression+of+metallothionein-1+gene+cluster+in+breast+cancer.">Genome-wide DNA methylation analysis reveals estrogen-mediated epigenetic repression of metallothionein-1 gene cluster in breast cancer.</a> Clin. Epigenetics. 7, 13 (2015).</li><li>Hsu, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter+hypomethylation+of+EpCAM-regulated+bone+morphogenetic+protein+gene+family+in+recurrent+endometrial+cancer.">Promoter hypomethylation of EpCAM-regulated bone morphogenetic protein gene family in recurrent endometrial cancer.</a> Clin. Cancer Res. 19, 6272-6285 (2013).</li><li>Wang, Y. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Distal+and+Genic+Methylation+in+the+Development+of+Prostate+Tumorigenesis+Revealed+by+Genome-wide+DNA+Methylation+Analysis.">Roles of Distal and Genic Methylation in the Development of Prostate Tumorigenesis Revealed by Genome-wide DNA Methylation Analysis.</a> Sci. Rep. , (2015).</li><li>Plongthongkum, N., Diep, D. H., Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+profiling+of+DNA+modifications:+cytosine+methylation+and+beyond.">Advances in the profiling of DNA modifications: cytosine methylation and beyond.</a> Nat Rev Gen. 15, 647-661 (2014).</li><li>Riebler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BayMeth:+improved+DNA+methylation+quantification+for+affinity+capture+sequencing+data+using+a+flexible+Bayesian+approach.">BayMeth: improved DNA methylation quantification for affinity capture sequencing data using a flexible Bayesian approach.</a> Genome Biol. 15, R35 (2014).</li></ol>

This protocol is developed in the laboratories of Dr. Tim Huang and Dr. Victor Jin at the University of Texas Health Science Center at San Antonio.

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...

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&#160;sequencing (MBDCap-seq), where we use an affinity enrichment technique to identify methylated regions of the DNA. This technique builds upon the methyl-binding ability of the MBD2 protein to enrich for the genomic DNA fragments containing methylated CpG sites. We utilize a commercial methylated DNA enrichment kit for the isolation of these methylated regions. Our laboratory has screened hundreds of patient samples using this technique and here we provide a comprehensive optimized protocol, which can be used to investigate large patient cohorts.
As evident with any next-generation sequencing technology, MBDCap-seq also requires a specific bioinformatics approach in order to accurately quantify the levels of methylation across the samples. There have been many recent studies in an effort to optimize the normalization and analysis process of the sequencing data9,10. In this protocol, we demonstrate one of these methods implementing a unique read recovery approach &#8212; LONUT &#8212; followed by linear normalization of each sample in order to enable unbiased comparisons across large number of patient samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Methylminer DNA enrichment Kit</td>
			<td>Invitrogen</td>
			<td>ME10025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 Streptavidin</td>
			<td>Invitrogen</td>
			<td>112-05D</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor Plus Sonication Device</td>
			<td>diagenode</td>
			<td>B01020001</td>
			<td></td>
		</tr>
		<tr>
			<td>3M sodium acetate pH 5.2</td>
			<td>Sigma</td>
			<td>S7899</td>
			<td>100ml</td>
		</tr>
		<tr>
			<td>SPRIworks Fragment Library System I</td>
			<td>Beckman Coulter</td>
			<td>A50100</td>
			<td>Fully automated library construction system</td>
		</tr>
		<tr>
			<td>Adapter Primers</td>
			<td>Bioo Scientific</td>
			<td>514104</td>
			<td>PCR primer mix</td>
		</tr>
		<tr>
			<td>Qubit</td>
			<td>Invitrogen</td>
			<td>Q32854</td>
			<td>Fluorometric Quantitation System</td>
		</tr>
		<tr>
			<td>PCR master mix</td>
			<td>KAPA scientific</td>
			<td>KK2621</td>
			<td>PCR master mix</td>
		</tr>
		<tr>
			<td>AMPure XP</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>PCR Purification beads</td>
		</tr>
		<tr>
			<td>EB Buffer</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 2000 Sequencing System</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

methyl-binding dna capture sequencing for patient tissues

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).

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...

Watch this Scientific Journal Video about Methyl-binding DNA capture Sequencing for Patient Tissues at JoVE.com

Methyl-binding DNA capture Sequencing for Patient Tissues

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 ...

The overall goal of this technique is to systematically investigate the genome-wide DNA methylation landscape in large patient cohorts. This method can help answer key questions in the epigenetic field, especially with regards to which regions in the DNA methylation landscape get deregulated in patients. The main advantage of this technique is large number of the patient can be investigated in a cost effective manner.This technology can have positive implications into the therapy and diagnosis of the patient because we can investigate existing biomarkers and also identify novel biomarkers. Though this method can provide insight into patient cohort, it can also be applied to other systems such as DNA methylation in cells derived from cultured cell lines from mouse-derived samples. Demonstrating this procedure along with Dr.Ya-Ting Hsu will be Mr.Joseph Liu, a technician in our lab.The sequencing part will be demonstrated by Ms.Dawn Garcia who is a technician in our sequencing core. After isolating genomic DNA from tumor or normal tissue according to the text protocol, add 1.2 micrograms of total genomic DNA to 96 microliters of TE buffer and 24 microliters of 5X bind wash buffer to make a 120 microliter solution. Sonicate the DNA solution for 30 seconds, then allow it to rest for 30 seconds.Repeat the sonication a total of 20 to 25 times. Use a bioanalyzer system with a commercial high-sensitivity DNA kit according to the manufacturer's protocol to run one microliter of sonicated DNA solution to check the size of the fragments. They should be in the range of 150 to 350 base pairs.To carry out single fraction elution, prepare elution buffer by making a one-to-one solution of low-salt buffer and high-salt buffer. Use 200 microliters of the elution buffer to resuspend the methyl-binding DNA or MBD beads and incubate the suspension on a rotating mixer for three minutes. After the incubation, place the tube on the magnetic rack for one minute, then carefully pipette the liquid without touching the tip of the pipette on the beads and transfer it to a clean DNase-free 1.5 milliliter microcentrifuge tube.Store this sample on ice. Repeat the elution for the second sample and collect it in the same tube as the first sample. To each non-captured wash and dilution fraction, add one microliter of glycogen, one-tenth the volume of three molar sodium acetate, pH 5.2, and two sample volumes of 100%ethanol.After mixing the solution well, incubate at negative 80 degrees Celsius for at least two hours. Then centrifuge the reaction at 11, 363 times g and four degrees Celsius for 15 minutes. Carefully discard the supernatant without disturbing the pellet and add 500 microliters of cold 70%ethanol.Spin the tube again before repeating the ethanol wash. Air dry the pellet for five minutes. Then use DNase-free water or buffer to resuspend it.Check that the total amount of DNA is over 20 nanograms according to the text protocol. Then place the DNA on ice or store at negative 20 degrees Celsius until further use. For DNA sequencing library preparation, use a fully automated library construction system and follow the standard protocol supplied by the manufacturer.Select DNA fragments of 200 to 400 base pairs in length for library preparation. After preparing the DNA sequencing library, remove the reaction and use a fluorometric quantitation system to quantify the DNA sequencing library. To set up a PCR amplification reaction, in a PCR tube add 15 microliters of DNA sequencing library, 25 microliters of PCR master mix, two microliters of PCR primer mix, and eight microliters of water.Use the program outlined in the text protocol, making adjustments as necessary based on the recommendations of the PCR mix manufacturer. Carry out enough cycles to produce two to 10 millimolar of library DNA. After cleaning up the PCR reactions, barcode each sample and pull four samples together into one lane of a flow cell.Use the standard 50 base pair single-read protocol of our sequencing. Carry out bioinformatics according to the text protocol. Using MBD cap seek to identify CpG islands across different genomic regions that are differentially methylated in tumor with respect to normal tissue, this study revealed that out of the total CpG islands located in gene promoters, 19.5%showed differential methylation in tumors compared to normal.Similarly, 55.2%of intragenic CpG islands investigated showed differential methylation. Intragenic promoters showed 28.1%And for gene promoters without CpG islands, 1.8%showed differential methylation. This heat map clearly depicts differentially methylated regions across 77 breast tumors, 10 breast normal tissues, and 38 breast cancer cell lines.In this figure, methylation is quantified in two endometrial cancer subgroups, non-recurrent vs. recurrent, in two patients at different CpG sites in the promoter CpG island of the identified target gene SFRP1. Once mastered, this technique can be done in four to five days if it is performed properly.While attempting this procedure, it is important to remember to first test the quality of a genomic DNA being tested. Following this procedure, additional methods like pyrosequencing can be performed in order to answer questions like, which are the exact CpGs in the identified differentially enriched regions that have been altered? After its development, this technique paved the way for researchers in the field of epigenetics to explore DNA methylation in large patient cohorts.After watching this video, you should have a good understanding of how to investigate genome-wide DNA methylation in patient samples using the MBD cap sequencing technique. Keep this in mind, sometimes it can be difficult to catch the high quality of the DNA from such as FFP and DNA samples, so proper assessment of the genomic DNA is always taken when we perform the procedure.

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8.5K Görüntüleme.  University of Texas Health Science Center at San Antonio. The overall goal of this technique is to systematically investigate the genome-wide DNA methylation landscape in large patient cohorts. This method can help answer key questions in the epigenetic field, especially with regards to which regions in the DNA methylation landscape get deregulated in patients. The main advantage of this technique is large number of the patient can be investigated in a cost effective manner.This technology can have positive implications into the therapy and d...