Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-6-12
Jump to navigation
Jump to search
sci-Methyl Seq Barcode 1 Design v3; Barcode 2 Design v2[edit]
Background[edit]
- Based on previous tests, it looks as though the original plan of second strand synthesis with an annealed ssDNA Adapter 2 doesn't work very well (it creates many unexpected band sizes). Consequently, in order to address this challenge, we want to redesign how we add Adapter 2 to our DNA fragments.
- We propose the following experimental pipeline, which we will be testing in parallel with the original annealing/second strand synthesis protocol:
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'
Barcode 1 Adapter Design (v3)[edit]
- We want to add an additional CC sequence to the 3' end of the top strand, while we can keep the same Adpt1_v2_comp as below:
Adpt1_v3 5' /5Phos/TTAGAGGTGGTCCCTCCTACCCGGCGTTTCC 3' Adpt1_v2_comp 3' TAATCTCCACCAGGGAGGATGGGCCGCAAA 5'
- NOTE: We only need to order Adpt1_v3 and we will just use the same Adpt1_v2_comp as before
Barcode 2 Adapter Design (v2)[edit]
- We likewise need to redesign Adpt2 sequence. We will keep mostly the same design as <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-4-28> but we will change the following:
- Add a complementary GG sequence to the bottom strand such that it Adpt2 will be able to bind and ligate to the sticky end formed after Adpt1 ligation
- Remove the complementary Filler 2 sequence from the adapter since that is no longer necessary for annealing (instead, the GG sequence will be enough to anneal to the sticky end)
- Make sure that the top strand (i.e. Filler 3, Barcode, and UMI), which is ligated onto the DNA fragment, contains no C's. Otherwise, that may be potentially converted to G's during bisulfite conversion.
Adpt2_v2 5' /5Phos/[GATAGG]TGGAGAGGGGAGAGGAGGTAAGGAGAGG 3' Adpt2_v2_comp 3' GG[CTATCC]ACCTCTCCCCTCTCCTCCATTCCTCTCC 5'
- We can use the following sequence to perform PCR in order to double check that Adtp2_v2 was added onto the DNA fragment correctly:
Adpt2_v2_PCR 5' CCTCTCCTTACCTCCTCTCCCCTCTCCA 3' <- This will anneal onto the 3' end of Adpt2_v2 (now includes Filler 3 and UMI sequence in order to increase melting temperature of fragment for PCR) and polymerize across the insert as follows: 5' -----[Barcode1]DDAA-----T|-----A|-----TTDD[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3' <------------- 5' We want to make sure that polymerization ends at the end of Filler2 from Adpt1 instead of continuing on to include the reverse complementary sequence of Filler 3