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* Methylation haplotyping - define the computational problem clearly. Send my drafts to Dr Zhang to look over. * Methylation LD - Add more normal tissues data, LMRs, overlap regions with cancer LD blocks * oxRRBS/oxBSPP/oxSelector for 5hmC quantification - order another oxBS-seq kit? - can't find the number for 6 reactions kit ($1000), 24 reactions kit is $3000. == April 3rd == * Last meeting: **First test to see if the control dna is damaged by doing ox and bs & then run a gel. **DNA damage fixing kit from NEB. **DNA Smart Kit. **Haplotype block analysis **Extend blocks to MONOD samples ===New results=== *oxidation bisulfite conversion treatment does fragment or degrade DNA, we yielded less DNA from oxBS than from BS alone. However, the treatment did not degrade 5hmC containing amplicons the most (5hmC - >70%, 5mC = ~90%, C = 37%). We were still able to see the amplicons on the gel. Oxidation bisulfite conversion did damaged the DNA and treatment with the PreCR Repair Mix from NEB appears to help. Although converted DNA are single stranded, some of the enzymes were still able to repair problems. **BY HOW MUCH? **How does the repair worked? what can cause problem in polymerization, what problems do oxidation damage cause. Taq DNA ligase, Endonuclease IV, Bst DNA Polymerase, Fpg, Uracil DNA Glycosylase (UDG), T4 PDG (T4 Endonuclease V) and Endonuclease VIII. **BS-seq appears to bias towards methylated DNA, oxBS-seq have even stronger bias toward methylated DNA because 5mC amplicons amplified better. BY HOW MUCH? (This may be a big problem, should move on to TAB-seq) ===Next steps=== *The amplicons that I used in the previous test did not have adaptors added. I will perform the digestion qPCR test on these to check the conversion efficiencies *I plan to repeat the control test one more time: titrate the amount of each modified DNA control added: 5hmC control > C control > 5mC control. I will also compare Epitect versus Zymo EZ methylation lightning kit. I just need to show that I can generate sequencing libraries from the 5hmC control DNA. I plan to do capture using HOTSPOTS460K probe: previously I had the problem of mapping rate being low, I just need to improve the mapping rate. **summarize all the experiments with optimizing HOTSPOTS460K. Repeat capture to test whether capture using all the probes or one subset will be different. sequence a small amount to assess the mapping rates, etc. *I plan to download and run the MAB-seq, TAB-seq, oxBS-seq data through my pipeline. Need to think about how to analyze the data. == April 24th, 2015== * MDS samples: compared Tet2 versus no Tet2 mutations, found more sites hypermethylated in Tet2 mutation patients compared to no Tet2 mutations. Need to label heatmap plot better. Clarify which one have the mutation and which one don't, number of sites in each group. p value. * 5hmC quantification: buy the Epimark Kit & use their control DNA instead. Move on with capture if successful with Epimark controls. * == May 1st, 2015 == * Confirmed with Dr. Zhang that we can process more MDS samples form Dr. Bejar * Need to move forward with capture using HOTSPOTS probes ASAP. I just need to process about 20 samples with HOTSPOTS. I realize that the collaborator might be uncomfortable moving forward with the commercial 5hmC controls at $20/sample because they are planning to assay 10s of samples. But I need to move forward faster with my HOTSPOTS probes capture on differentiation samples. * I have finished mapping the Salk's Human Tissue WGBS data and will be copying them to genome-miner. * I have gotten the Tet proteins from NEB, and I need to perform side by side comparison, perhaps with H1 ESCs so I can compare across different technologies and published datasets. Since we are not going to perform whole genome sequencing with these samples, I will need to capture with my HOTSPOTS probes/or DMR330K first. == May 22nd, 2015 == * order the IDT control oligo - [[File:DD15_idt_dna_epimark_ctrl_order.png | 600px]] * contact Nature Protocol papers authors to ask about the controls - contacted Dr. Balasubramanian from the oxBS paper. ** Dr. Balasubramanian: "If you followed the protocol exactly, 5hmC should undergo very little conversion to U. Supplies of d5hmCTPs are often contaminated with some dCTP which gets incorporated much faster than d5hmCTP itself during the PCR - so this is worth checking out (ideally by LC-MS analysis). You might also wish to try the commercial oxBS kits, now provided by CEGX (http://www.cambridge-epigenetix.com/) who have optimised the formulations, workflow and controls to make them even more robust (we actually used their oxidant formulation in the Nat protocols paper). Best wishes for your experiments." * put the BSPP dual index design on wiki: fixed the sequencing primer designs from last time, http://genome-tech.ucsd.edu/LabNotes/index.php/Illumina_GA/Oligo_info#Library_Free_Multiplexing_Primers_for_Dual-Index * use H1 for TAB experiment comparing ngTet, mTet2, and Wisegene mTet1: would Dr. Bejar & Brian be able to share with us his CRISPR plasmids so we can do Tet2 and DNMT3a knockout on H1 and then differentiate towards definitive endoderm? == May 29, 2015 == * Can we get the knockout cell line for Tet2 in hESCs? Look at the Meissner paper on DNMT3a knockout. Would Tet2 have an effect on 5hmC? * Perform qPCR on bisulfite converted controls. Which polymerase would have the best efficiency when amplifying DNA containing CMS? - HemoKlentaq, Phusion U, PfuTurbo Cx? Use the same sequence with no modification as control. * What if instead of 5hmC, we have 5ghmC (for TAB-seq)? *
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