Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-6-13
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sci-Methyl Seq Barcode 1 Design v3; Barcode 2 Design v2
Background
- Just yesterday, we designed new sci-Methyl Seq Adapter 1 and Adapter 2 sequences and ordered them to test whether we could ligate on Adapter 2 instead of using the current annealing method. However, looking further into the protocol, it would be prudent to begin designing Adapter 1 such that it will be appropriate for bisulfite conversion and PCR. In order to do this, we need to do the following:
- Adjust Filler 2 such that it does not contain any G's, which would result in C's in the final sequence that may be bisulfite converted (making PCR inefficient/impossible as primers need a consistent binding site for adding the P5/P7 adapter regions)
- Begin testing the formation of dsDNA Adpt1/Adpt2. This would require using a method similar to the HpyCH4III digestion outlined here <http://www.nature.com/nprot/journal/v9/n11/box/nprot.2014.170_BX1.html> to create the T-tailed Adpt1. dsDNA Adpt2 can be created just by using polymerization/end-repair.
- Create final PCR primers that will add the P5/P7 sequencing adapters to both ends of the DNA fragment
Overview
- Below is a general overview of the experimental procedure to ligate Adpt1 and Adpt2 (same as <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-6-12> but with PCR added in)
End Repair/dA-Tailing 5' -----A 3' 3' A----- 5' | V Add Adpt1_v3: 5' /5Phos/-----TTDD[Barcode1]-----CC 3' 3' T-----AADD[Barcode1]----- 5' | V Ligate Adpt1_v3 (using same optimized ligation protocol as before) 5' -----[Barcode1]DDAA-----T|-----A|-----TTDD[Barcode1]-----CC 3' 3' CC-----[Barcode1]DDTT-----|A-----|T-----AADD[Barcode1]----- 5' | V Add Adpt2_v2: 5' /5Phos/-----[Barcode2]DDDDDDDD----- 3' 3' GG-----[Barcode2]DDDDDDDD----- 5' | V Ligate Adpt2_v2 (using same optimized ligation protocol as before) Nick V 5' -----DDDDDDDD[Barcode2]-----GG|-----[Barcode1]DDAA-----T|-----A|-----TTDD[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3' 3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]DDTT-----|A-----|T-----AADD[Barcode1]-----|GG-----[Barcode2]DDDDDDDD----- 5' ^ Nick | V Denature/separate fragments (will break up DNA at nicks) 5' -----[Barcode1]DDAA-----T|-----A|-----TTDD[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3' 3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]DDTT-----|A-----|T-----AADD[Barcode1]----- 5' | V Bisulfite conversion | V PCR, 1st cycle (Add P7 sequencing adapters) P5: 5' AATGATACGGCGACCACCGA 3' P7: 5' CAAGCAGAAGACGGCATACGAGAT 3' 5' -----[Barcode1]DDAA-----T|-----A|-----TTDD[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3' 3' <----- \ P7 5' 5' P7 \ -----> 3' 3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]DDTT-----|A-----|T-----AADD[Barcode1]----- 5' | V PCR, 2nd cycle (Add P5 sequencing adapters) 5' P5 \ -----> 3' 3' -----[Barcode1]DDTT-----A|-----T|-----AADD[Barcode1]-----GG|-----[Barcode2]DDDDDDDD-----P7 5' 5' P7-----DDDDDDDD[Barcode2]-----|GG-----[Barcode1]DDAA-----|T-----|A-----TTDD[Barcode1]----- 3' 3' <----- \ P5 5'