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== 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.
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