Questions related to DNA Methylation
We are measuring methylation at the POMC gene on human DNA.
We are getting good results with out PCR but are consistently having issues with low peak height and baseline drift on the sequencing step.
The option appears to be to increase PCR product though the manual says the maximum is 10ul. We have tried 15ul and this shows some improvement but does not resolve the issue entirely.
Does anyone have experience of the machine and can you put more product in?
Any suggestions much appreciated.
I have been collecting DNA methylation data for TNBC patients and while I've only been using data produced using Illumina HumanMethylation450 BeadChip the methylation values vary greatly. To be more specific some of the datasets contain values that only range from 0 to 1 while others have values that range from -5 to 7.
I am unsure if the content of the data is completely different or if the data that ranges from 0 to 1 has just been transformed using something similar to a sigmoid function. Any help would be greatly appreciated.
I am trying to investigate the global methylation with the kit Imprint Methylated DNA Quantification kit (Sigma-Aldrich). However, the results of all my samples were higher than my control. Thus, all samples showed more than 100% of methylation.
Is anyone with the same problem?
We tried different concentrations of DNA but we always found the same: DNA methylation was higher in our samples compared to the methylated control.
In many instances, certain DNA methylation activities can be harmful to humans, causing cancers and various disorders. A majority of my research about plant DNA methylation however shows mainly beneficial effects to the plant when the plant is treated with a stressor. Can methylation also be harmful to a plant a lot?
I'm trying to predict age using DNA methylation but I'm not getting the expected amplicon size on the gel image. Please any suggestions?
Centers in India that offer hands-on training in bisulfite conversion, DNA methylation, expression, and epigenetics are preferred.
good day! I am a currently researching about epigenetic variations (Mainly on DNA methylation) of a fish that can change habitats (from saltwater to freshwater), my main goal/topic is about osmoregulation and my target tissue is gills. my original plan was to get samples from both habitats regardless of what sex of the fish is. however, most of the literatures that I have been reading collected male only fish. I would be very grateful for your insights.
Is it possible that a monoclonal cell line expressing gfp in a stable way can lose or decrease its expression in the course of the passaging (example: after 20 passaging).
Because of for example a methylation of the promoter etc.?
Do any of the cells have genomic DNA that is 0% methylated? I found scientific reports indicating that peripheral blood mononuclear cells (PBMCs) may exhibit such properties. In my research, I use bovine material and I would like to make a curve for Methylation-Sensitive High Resolution Melting (MS-HRM) analyses by mixing 0% and 100% methylated DNA. Unfortunately, such DNA for cattle is not commercially available. I think, it will be more difficult to obtain 100% methylated genomic DNA.
Hi, I'm doing bisulfite sequencing to study DNA methylation status of my target gene.
I'm using QIAamp DNA Mini kit(QIAGEN) for obtaining gDNA samples and EZ DNA Methylation-Lightning Kit(Zymo Research) for bisulfite conversion.
As protocol from zymo said that 200~500ng gDNA 20ul is optimal condition for bisulfite conversion, I'm using 200~500ng gDNA(sometime 500ng if my gDNA concentration is enough, but sometime I use around 200~300ng if my gDNA concentration is low.)
My problem is I think I'm getting unexpected results.
In my lab I'm the first person to study DNA methylation and use such kind of kits, I'm trying to get same, or similar results from previously published paper before I get results of my target gene.
The gene used in the paper was highly methylated and methylation status of each CpG sites were around 60~90%(mostly 70, 80%)
But when I did sequencing, only 50% were methylated.
Also CpG sites from each colony was all methylated or all unmethylated.
(I mean, I tried sequencing from 8 colonies and results from 4 colonies said all of the CpG was methylated and results from rest of the colonies said all of the CpG was unmethylated-so I'm hard to trust my results.)
I don't think bisulfite reaction didn't work because almost of non-CpG cytosines was converted to uracil.
I think I'm doing something wrong with my expreiments, so I needs some advices.
1. Is gDNA around 200~300ng not sufficient to perform bisulfite conversion? I've heard that too less gDNA amount or too much bisulfite treating could cause conversion of methylated cytosin to uracil. If it's not sufficient, how much would be optimal?
2. Is it common to get only all-or-none methylated result as me?
3. I didn't use RNaseA during extracting gDNA so maybe some RNA will be remaining in my gDNA. Would this be big problem?
4. Are there some other factors that could be a cause of unsuccessful or unreliable bisulfite conversion/sequencing? (There would be a lot, but I want to check if I'm missing something.)
If you have any other ideas or advices to perform successful bisulfite sequencing, I would like to know it whatever it is. Thank you for reading long question.
I am looking forward to determining the level of DNA methylation in cell lines and animal tissue samples. The determination of 5mC and 5hmC and DNMT1/3 are considerably important. What are the important differentiating steps for samples processing, for analysis of 5mC, 5hmC through HPLC UV/LCMS.
I need epigenetic data file run through the hovarth clock. DNA Methylation Age Calulator, but did not find any description how it should be done?
Please can you help me to find link to describe the process of running the data?
For example, DNA has functional groups such as methyl groups and nanoparticles can interact with them and alter them. I was wondering if we have any technique by which we can quantify them or maybe do a qualitative analysis. Any leads would be great, Thank :)
I have a bed file of DNA methylation, I wanted to know do we analyze sex chromosome data with other sites or not? Any help would be great.
DNA methylation represents a covalent bond between a methyl group and a cytosine. To capture the DNA methylation patterns at a certain point in time, is it sufficient to dry the plant tissue or is it necessary to flash-freeze it in liquid nitrogen?
Currently, I am working on an aggressive breast cancer cell line. I intend to restore and enhance the expression of tumor suppressor genes by siRNAs against the DNMT gene and induce apoptosis after treating the cells with siRNAs and subsequently exposed to a chemotherapy drug. I performed the MTT and Annexin V/PI apoptotic assays and I have got weird results.
In MTT assay the conc. of chemo drug with no siRNA was ranged from 100nM to 3200nM and the incubation time was 12 hours. In that experiment, the IC50 was 270nM. But, In cells treated with siRNA and the drug, even the highest conc. of the drug, i.e. 3200nM, did not induce cell death (the ODs were the same for each conc.). To clarify, I reverse transfected the cells with siRNA using RNAimax lipofectamine by Thermo fisher protocol (12 hours incubation time). subsequently, replaced the medium with the serially diluted conc. of the drug in RPMI containing FBS. after 12 hours of incubation, I have proceeded with MTT assay.
In the annexin V/PI experiment, I reverse transfected the cell only with the siRNA. the percentage of the cells in the Q4 quadrant was 90%!! As I understand, the siRNA against DNMT is supposed to induced apoptosis. but the results are showing a completely different outcome.
I have done the same procedure on CMML cells and the results were showing a considerable induction of apoptosis. the only difference was the transfection time, 19 hours against 12 hours.
It is known that DNA methylation in promoter regions containing CpG islands is responsible for downregulation of genes. How about genes which don´t contain CpG islands in their promoter region? I have been investigating a gene which contains CGs in its promoter region. Based on data from Xenabrowser, there is a negative correlation between methylation and expression levels. However, MethPrimer didn´t identify a CpG island (based on criteria such as GC% content or O/E ratio) in that region. How likely is it that methylation plays a role in the regulation of this gene?
Thank you very much!
We know that epigenetic factors are denoted by alterations in DNA methylation, histone modification abd transcription of regulatory non coding RNA such as microRNAd but are there additional epigenetic changes that take place that do NOT involve these mechanisms?
I tried to run this function sitetest to perform Site-level Differential Methylation Analysis using IMA package but I got error message.
sitetestALL = sitetest(dataf,gcase="KO",gcontrol="WT",testmethod ="wilcox" ,Padj="BH", rawpcut = NULL,adjustpcut =NULL,betadiffcut = NULL,paired = FALSE) and I got this error message: Error in wilcox.test.default(x[1:length(lev1)], x[(length(lev1) + 1):(length(lev1) + : not enough (finite) 'x’ observations
Can you help me to solve this problem?
I have an inducible expression of the enzyme in a newly derived bacterial strain, and methylation may be affected. I want to test methylation efficiency with a bisulfite kit. The kit is the EZ DNA Methylation kit (Zymo Research, D5001).
Do I need to make a special plasmid with a certain sequence for the bisulfite test, or it works more or less equally for any DNA sequence?
I am looking for an online tool to see whether a certain methylated CpG site can affect the expression of a certain gene or not?
I am trying to predict age from DNA methylation. I am using conventional PCR master mix, but I didn't get my PCR product. Is it due to Master Mix? Should I use HotStar Taq Master Mix ?
I use pyridine borane to react with DNA. I observed that DNA recovery on the purification stage after pyridine borane reaction is very low, at 10%. Therefore, I would like to kindly ask you about the following:
Does pyridine borane fragment genomic DNA?
Does it affect the efficiency of DNA binding to the column bed during purification?
Does anyone recommend a suitable purification method and tested binding buffers or other suggestions to improve DNA recovery?
Additionally, I have a question, the pyridine borane solution with DNA is shaken for 16h 850rpm, but I wonder if I can mix with vortex before incubation for better distribution of the substance in the solution?
I will be grateful for all suggestions.
I am a relatively new in DNA methylation field. I am interested in DNA methylation status of some CpGs sites: two from exons and one from introne. I would like to monitor their methylation status without killing the cells, so conventional sequencing approach do not work. I found the so-called Reporter of Genomic Methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein (Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution https://www.cell.com/cell/references/S0092-8674(15)01098-3 ).
Can someone please explain will the following work or not and why? I plan to make AAV vector with 100-200 bp with the CpG of interest + RGM. These bp also have additional methylated-capable CpGs which correlate with methylation status of the CpG of interest, so if it will report the others it is also fine.
I will appreciate every suggestions or references
I'm doing an MSc with the Open University researching DNA methylation (5mC and 5hmC) in relation to Alzheimer's disease. I've no connections with a lab, and working from home I am trying to get to grips with the technologies/platforms that are mentioned in the papers I am reading. Could anyone explain the difference between BS and 450K, and if there are benefits in using both in the same study, or is one superior to the other?
Dear all, I´m experienced in analyzing datasets of gene expression, but for my new project I don´t seem to find a dataset with different time points that is fundamental for my objective. I did find a dataset of an experiment that as the perfect design of what I need but is of data from DNA methylation. It is possible to identify MicroRNAs that putatively are differential expressed based in their methylation state?
Thanks in advanced.
How can one report the result of an assay on global DNA methylation done using an ELISA based technique.
Hi, I treated the cells with 5-Aza-dc and TSA and analyze a specific gene transcription and found out that the gene was induced due to both treatment(q-PCR) relative to control. Then I preform a western blot and found out that the protein level was reduced. This kind of dis-correlation can happen with DNA methylation and histone actylation inhibitors? can someone give a reference for this kind of dis-correlation?
I'm trying to analyse the methylation status of the promoter of my gene of interest. I run a few tests to make sure everything is working fine. However, one of my problems is the methylated DNA control shows a methylation percentage of 79-87% range, when it should be 100%.
I think this could be due to some noise, as the non-methylated peak are 5-6 vs 35-50 high of the methylated peaks. Also, the control bases (not present in the sequence) have a tiny peak of 2-5 high, which is clearly noise.
I've been looking around the software but can't seem to find a parameter to set up a noise line so those peaks don't get 'identified' as non-methylated. Is there such a thing? What could I do otherwise?
Note. I know my methylated control is ok as HpaII digestion got a single band compared to a smear of the non-methylated controls. Also, I run methylated DNA that I BS converted and a control that I bought already converted, and both show similar ranges of methylation percentage (always lower than 90%).
Many thanks for your help,
When we want to study epigenetic effects of bush/wild fires, using an ongoing cohort, we need to focus on a limited list of CpGs. Although necessary, the funding agency does not allow exploratory research. We are considering to include CpGs that are differentially methylation after exposure to smoking (568 CpGs), to PM10 and PM2.5 exposure (~70CpGs), and to stress (?? CpGs).
However, we need a smaller list.
Any preliminary data is welcome.
I work with whole genome bisulfite sequencing and I would like to validate the biological response from DNA methylation regions with gene expression.
My question is about number of samples.
I planned work with 4/6 differents genes and 15/20 brain tissue samples.
My samples are very difficult to get, and I'm asking myself if 15/20 sample are too few for a biologial response validation.
If someone could help with that, I would be very thankful.
am gonna to study the methylation status for target promoter gene . I have seen that there are alot of kits can be used such as Zymo kits and Qiagen kits but which one is the best ,also in each one there are different kits . So which one exactly is best for me for studying the DNA from blood samples ?
Also if you kow there is an OPTI-Bisulfite method ? Any one used it ?
recently, I plan to check the influence of DNA methylation and methylation levels on protein activities in vitro (a protein with both methyl-DNA and unmethyl-DNA binding properties), So I need first try to amplify a specific fragment with different methylation levels.
for an example, 50% methylation levels, each DNA molecule is methylated, and 50% cytosine is replaced by methyl-cytosine occasionally, rather than 50% DNA molecules are methylated, 50% unmethylated (which I can acquire by mix unmethylated and methylated DNA)
As I consumed, If I mix different ratio of dCTP:dmCTP for PCR mixtures (50%), during PCR, 1/2 of cytosine (C) position will be replaced by methyl-cytosine (mC) accasionally. the final products will contain 50% mC.
after, I need to check if the DNA contains different levels mC. the slot blot is used, and anti-5mC antibody is applied to detect the methylation levels.
the unthylated PCR product (no dmCTP) show no signal, while the 100% methylated DNA (no dCTP) show strong signal. but the PCR products amplified with different dCTP:dmCTP ratio show similiar signals (10%, 25%, 50%, 75%) to 100% methylated DNA.
could I conclude that all PCR products, even the one with only 10% dmCTP added, are 100% methylated, all C is replaced by mC.
if not, which methods should be applied to detect the methylation levels.
Hello everybody, I am new with the meta-analysis in Genome Wide Data so I have this doubt. I have read METAL documentation, which is by far the most used meta-analysis software in both EWAS and GWAS microarray data, but I cannot figure out how would be the input for EWAS analysis. As METAL was originally designed for GWAS, one of the inputs is to provide both the reference and no reference allele. Therefore as EWAS arrays do not rely in allele frequencies but in a quantitative measure, I would like to know how would be the input in METAL regarding this case. Thank you so much in advance for answering this issue (which may be easy, but I certainly do not know)
I am doing the age related methylation study of snow leopard. Since there is no commercial kit for me to use as control DNA, I made "fully methylated" by M.sssI with both blood DNA and its WGA product.
For blood DNA, I followed the protocol and incubated for 1hr. For WGA product, I tried 2 types: 1st, just incubating it for 4hr; 2nd, incubating it for 2hr and then purifying the product and replenishing reagents and incubating for another 2 hr.
However, according to the result I got from MS-HRM (see in the attachment), they should different relative signals which implied to different methylation rate (100% blood DNA 1hr > 100% WGA 2hr→purification→2hr > 100% WGA 4hr).
I am wandering whether it is because of the insufficient incubation time of WGA or the poor quality of WGA. I also want to know how to check whether the methyation rate really reached 100% or not. If not, how to improve it.
I am also considering to use synthesized 100% methylated DNA of target region if making 100% methylated DNA from M.sssI is too hard. Since the concentration of target region of synthesized short DNA strands must be higher than normal DNA, I think I need to adjust the input amount of synthesized DNA carefully before apply it. Does anyone has the experience, especially when using MS-HRM?
Hello, Does any one faced transgene silencing after transfection? I have been trying to get good expression from a recombinant antibody molecules cloned in pUC based vector and transfected in CHO cells. Although the cells were selected and checked for expression the expression is very less and there is no reporter gene available to check the transfection efficiency.
I have recently come across the insulation of the transgene on either side with insulator sequences. so did anyone tried the same? If so can i get any protocol or any paper link to learn how to include insulators for achieving high level of transgene expression?
Thanks in advance.
I have a list of genomic coordinates from differentially methylation regions and a I need to extract from them a list of genes, and I don't want do look one region by one.
Is there a R package or other bioinformatic tool that can help me?
Usually when we discuss about epigenetic modifications specifically DNA methylation, we observe hypermethylation of promotor or CpG usually results in gene silensing/ low regulation.
In my case promotor region of this specific gene is hypermethylated but mRNA levels and protein levels are upregulated which results in increased in metastasis in breast cancer.
I am wondering what technology I should use best for the detection of methylated DNA. More specifically, I am investigating the MLH1 gene and was thinking of using either MS-HRM, pyrosequencing, qMSP, MSRE analysis. What would you recommend in term of convenience, efficiency and price?
Many thanks :)
Our lab has an abundance of ethanol preserved tadpoles and we were wondering whether or not there would be troubles getting typical bisulfite sequencing data from these samples. I understand the methyl group is covalently bound to the DNA and this might make it robust against degradation in the short term (~10-20 years). Any thoughts or advice would be appreciated!
Recent Litterature is little poor concerning DNA extraction and possible possible oxidation occuring during extraction. Some works give some protocols to prevent formation of 8oxodGuanosine while the extraction but oxidation of phenol for example could have an impact also of MetCytosine and change rate of MetC / OhMetC / foMetC and cacMetC.
If someone has tips to extract DNA in order to measure DNA methylation and 8oxodG representing the 'in vivo reality'...thanks in advance !
I have analyzed two DNA methylation data sets:
1) Illumina EPIC
2) Hiseq2500 bisulphite sequencing
I selected one CpG site from the Illumina EPIC analysis and extracted its genomic coordinate on Hg19. For comparison, I want to find the same CpG site in the bisulphite sequencing data set on Hg38. Is this possible?
More details, I tried to look up the exact DNA base, with no luck (made sure to match Hg19 and Hg38 coordinates). I also search in a window +/- some bases around the DNA coordinate, with no luck.
Can it be that the bisulphite sequencing data set simple did not have enough cover? Or is it something else I may have overlooked?
Hi scientists.... recently I give a treatment to primary fibroblast, and I found that the number of 5mc stained punctum decreased significantly. Anyone have some experience or knowledges to explain this? Thanks!
Dear community members,
We have available WGBS data for a species. We used Bismark to map the reads to the reference genome. Unmethylated lambda phage DNA was spiked-in, to estimate the bisulfite conversion error rate. My question is, how can I use the information of this error rate to distinguish truly methylated reads from false positives?
I have seen that many people use a binomial distribution to achieve this, and apply a Benjamin-Hochberg correction citing their 1995 paper (https://www.jstor.org/stable/2346101?seq=1#page_scan_tab_contents).
Is there a tool or an easy way to do this? Excuse my ignorance but I am really new to the field.
Thank you for your time,
I have whole genome bisulfite sequencing (WGBS) data for a species without reference genome. Is there any pipeline that I can use to process my data ?
What I am looking for are studies related to parents' ACES leading to autism in their children via changes in DNA; i.e. DNA methylation.
We have the link from ACES to DNA methylation
We have the link from DNA methylation in the parent to autism in their child https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856164/
However, I have not seen any study going from parent's ACES to children's autism. It might be there. I just have not seen it. Thanks.
I am doing bisulfite treatment on DNA extracted from rat brain and I want to know the treatment has been done completely or no. what controls can I use for that ? Can I use DNA extracted from rat lymphocytes as a fully unmethylated DNA? Can I make fully methylated DNA by treatment a normal DNA extracted with M.SssI enzyme?
thanks in advance
I know phenotypic differences but now I am interested to know the differences at genetic level e.g. Indels or DNA methylation. Thank you
Hello everybody, I am working in a conifer tree species, initially i need to propagate the plant through callus, i have established the callus induction but i struggle to regenerate the plant through callus, so i have planned to regenerate the plant through epigenetic remodeling. can we regenerate the plant through DNA methylation process..? i looked at the protocol for this. i would appreciate if anyone have experience in the plant regeneration via epigenetics. Thank you.
thank you in advance for your help.
Can isohelix genefix be used as a collection method to assess methylation levels in DNA from human saliva?
I.e. would it be ok to keep the saliva at room temperature in the hisohelix genefix buffer for several days? Will this affect the methylation status of the DNA or will DNA methylation be stable under these conditions?
Thank you immensely for your help,
Dr. Antonio Inserra
I am trying to quantitate promoter methylation of four genes ( SALL3, FERD3L, BARHL2 and PHOX2A) in breast cancer tissue sample and breast cancer cell lines. Can anyone suggest me which is the best method for quantitation of DNA promoter methylation?
Previously, I noticed that some protein harbor differential phosphorylated site could distinguish the cancer stage during the cancer development. Similarly, I am wondering the different methylated CG sites at the promoter or CpG island may also present different stages of the cancer. The methylation value of the probes in the same gene is different, which is consistent with the definition of Differential Methylated Regions(DMR). So, here is my question: can we find some specific methylated CGs or probes in the same gene to distinguish the cancer type or the stage? Then we can use the simple probe-based RT-PCR or to find out the cancer and its type or stage.
Due to that the DNA methylation and the RNA methylation could both regulate the gene expression, so here is my confusion: does the DNA methylation have the effect on RNA methylation? For example, the gene CDO1 is hypermethylated at the CpG island in LUAD, dose this DNA hypermethylation effect the RNA methylation pattern or level? By the way, these two kinds of methylation has the same effect on gene expression or not? And which is the key regulator to the gene expression?
I want to see global DNA methylation status of samples. What are the different ways by which i can check this?
After doing bisulphite conversion, what all genes i can sequence to understand level of global DNA methylation?
Hi, I'm going to research on mechanisms by which cells differentiate in different directions at the epigenetic level. And I choose WGBS to study DNA methylation. Do you have any suggestions on what does the WGBS data sue for, or any research ideas on my project, or any related articles？Thanks!!!
I'm studying a gene whose expression is regulated by DNA methylation. Cancer cell lines that express this gene show strong hypomethylation, while cells lines that don't express it show hypermethylation. It's also from a family of genes (LY6 family) that are nearly all regulated by DNA methylation. I've confirmed this methylation profile with bisulfite sequencing.
In cells that don't normally express it (hypermethylated cells), I see a strong (~20 fold) increase in expression following radiation treatment. I hypothesized that this was due to changes in methylation. But when I did bisulfite sequencing on irradiated cells, I see that they are still methylated.
Is there any mechanism (besides methylation) that can explain this? I'd really appreciate any help! Thanks!
I am working with DNA methylation currently. I am creating hypomethylation condition in the cells. so how to confirm that the hypomethylation has occurred.
sequencing of only LINE-1 gene will be sufficient to see the global methylation?
or what are the different methods to study global DNA methylation?
I am trying to monitor the methylation statuses of a certain set of genetic loci in glioblastoma cells following a treatment protocol. Can anyone please suggest some easy-to-use assay kits to analyze the methylation levels at certain genetic loci?
So these few days I was working with ACTIVE MOTIF Global DNA Methylation LINE-1 Kit, but I have no results because standard curve has a weak signal. For example, the level of "Standard 0" should be at least 0.300 - 0.450 but my result was 0.249. Level of "Standard 100" should be 1,300 - 1,900 but my result was 0,552. I tried it a few times and results I get is almost the same. I also tried to troubleshoot it with Instruction Manual but it didn't help me.
I am searching the literature for an article about DNA methylation signatures in breast cancer. I am finding a lot of articles showing comprehensive analysis between different subtypes of breast cancer, but I did not find one showing ALL the cpg sites that were differentially methylated in tumor versus normal-like breast tissues. If anybody can help, I will be grateful.
At the moment I am analysing the RNA-seq, ATAC-seq and Methylation datasets of the same mouse-derived cell-type. Methylation was done not as bisulfite sequencing, but as an enzymatic enrichment for DNA fragments (after sonication), containing methylated CpGs with subsequent DNA library prep. It results in ATAC-like peaky view on IGV, so I focus not on the particular cytosines, but on the overall hyper/hypomethylations of promoters, etc.
I have found, that in some cases the Methylation peaks follow the exon structure, and quite often the regions, which were methylated, were not accompanied with Tn5 accessibility on ATAC-seq.
Do you have an idea why introns are not methylated, in contrast to exons? Is it correct to say that the regions of chromatin, which are closed, tend (not?) to be methylated? Does DNA methylation decrease the Tn5 cutting efficensy?
I need to known a DNA sequence for a gene in the plant(V.faba) in order to design primer for PCR for DNA methylation purpose analysis.However I only had the mRNA seq and AA seq for the gene in this plant. What should I do
So, I have read quite a lot of papers that have studied the structure of DNMT3A and its mechanism; however, I couldn't really understand the mechanism fully. Hopefully, you guys can help me understand :)
Based on what I have read, DNMT3A works as a heterotetramer (DNMT3L-DNMT3A-DNMT3A-DNMT3L). Also, based on two papers published in Nature by J. Song and X. Cheng, the two DNMT3A active sites target CpG sites that are separated by approximately 10 to 14 base pairs apart.
So for example, if the DNA sequence is
5'-CATGCGTTCTAATTAGAACGCATG-3' (sense strand)
3'-GTACGCAAGATTAATCTTGCGTAC-5' (antisense strand)
One of DNMT3A active site will methylate CG (1) and the other DNMT3A active site will methylate CG (2).
Now my question is whether this DNMT3A methylates only the sense strand CG or only the antisense strand CG or both.
Also, if only one of my DNMT3A has a mutation which results in inactive form of DNMT3A (meaning formation of DNMT3L-inactive DNMT3A - active DNMT3A- DNMT3L), what will happen?
Should I expect either CG (1) or CG (2) methylated but not both?
We are planning to assess DNA methylation profile of 100-200 cells using bisulfite sequencing on the NextSeq500 instrument. However, the kits designed for single cells (Accel-NGS® Methyl-Seq DNA Library, Pico Methyl-Seq™ Library Prep Kit, QIAseq™ Ultralow Input Library Kit) produce genome-sized libraries that are better to sequence using more high-throughput platforms such as HiSeq or NovoSeq. Other solutions, e.g. TruSeq® Methyl Capture EPIC Library Prep Kit, generate enriched (not so big) libraries that can be sequenced using NextSeq but require a lot of input DNA.
Does anybody know solutions for methyl-seq library preparation and targeted enrichment using a low number of cells? Other words, we need a kit that is similar to TruSeq® Methyl Capture EPIC Library Prep Kit but is designed for a few cells.
Thank you in advance for your suggestions and opinions!
I am looking into using DNA methylation to test whether patients have accelerated aging.
I have seen researchers use Hannum's weights for CpGs in order to calculate accelerated aging in some patient populations.
How do we account for the standard error? Where can I find a pipeline for this calculation?
Dr. Magnus Nordborg recently pointed out in a presentation that is no evidence that methylation is involved in either development or adaptation and there is still little evidence of the involvement with gene regulation.
Also, the paper "Epigenomic Diversity in a Global Collection of Arabidopsis thaliana Accessions" (Kawakatsu et al. 2016) showed methylation in Arabidopsis strongly correlated with geography and climate of origin.
It seems that environment controls both methylation and gene expression but there is not necessarily a cause-effect relationship between these two.
I have DNA methylation data from the Illumina EPIC beadchip (information on 850 000 CpG site). Since SNPs can influence the methylation results, it is advised to filter CpG sites located close to SNPs. The question is how close.
I read that 10 base pairs is commonly used. But in the article of Zhou (2017, Comprehensive characterization, annotation and innovative use of Infinium DNA methylation BeadChip probes) it is noted that the 10 base pair distance is too stringent, since >50% of the EPIC probes carry SNP annotations with a distance < 10 bp. They advice to use a distance of 5 base pairs.
Do you agree with this?
I'm using entropy to measure dispersion in DNA methylation values.
However, I'm not sure which statistical test I may use to determine if the differences that I observe in entropy values between two groups are significant.
Thank you for your answers.