Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-6-12: Difference between revisions
Jump to navigation
Jump to search
>Cjwei (Created page with "=sci-Methyl Seq Barcode 1 Design v3= ==Background== *Based on previous tests, it looks as though the original plan of second strand synthesis with an annealed ssDNA Adapter 2...") |
>Cjwei No edit summary |
||
Line 1: | Line 1: | ||
=sci-Methyl Seq Barcode 1 Design v3= | =sci-Methyl Seq Barcode 1 Design v3; Barcode 2 Design v2= | ||
==Background== | ==Background== | ||
*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. | *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. | ||
Line 24: | Line 24: | ||
Nick | Nick | ||
V | V | ||
5' -----'''DDDDDDDD'''[Barcode2]-----GG|-----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----| | 5' -----'''DDDDDDDD'''[Barcode2]-----GG|-----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----CC|-----[''Barcode2'']'''''DDDDDDDD'''''----- 3' | ||
3' -----'''''DDDDDDDD'''''[''Barcode2'']-----CC | 3' -----'''''DDDDDDDD'''''[''Barcode2'']-----|CC-----[Barcode1]DDTT-----|A'''-----'''|T-----AA''DD''[''Barcode1'']-----|GG-----[Barcode2]'''DDDDDDDD'''----- 5' | ||
^ | ^ | ||
Nick | Nick | ||
Line 31: | Line 31: | ||
V | V | ||
Denature/separate fragments (will break up DNA at nicks) | Denature/separate fragments (will break up DNA at nicks) | ||
5' -----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----| | 5' -----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----CC|-----[''Barcode2'']'''''DDDDDDDD'''''----- 3' | ||
3' -----'''''DDDDDDDD'''''[''Barcode2'']-----CC | 3' -----'''''DDDDDDDD'''''[''Barcode2'']-----|CC-----[Barcode1]DDTT-----|A'''-----'''|T-----AA''DD''[''Barcode1'']----- 5' | ||
==Barcode 1 Adapter Design (v3)== | |||
*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' | |||
*<span style="color:red">'''NOTE: We only need to order Adpt1_v3 and we will just use the same Adpt1_v2_comp as before'''</span> | |||
==Barcode 2 Adapter Design (v2)== | |||
*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_v3 5' /5Phos/[GATAGG]'''TGGAGAGG'''GGAGAGGAGGTAAGGAGAGG 3' | |||
Adpt2_v3_comp 3' GG[CTATCC]'''ACCTCTCC'''CCTCTCCTCCATTCCTCTCC 5' |
Revision as of 15:27, 12 June 2017
sci-Methyl Seq Barcode 1 Design v3; Barcode 2 Design v2
Background
- 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)
- 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)
- 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_v3 5' /5Phos/[GATAGG]TGGAGAGGGGAGAGGAGGTAAGGAGAGG 3' Adpt2_v3_comp 3' GG[CTATCC]ACCTCTCCCCTCTCCTCCATTCCTCTCC 5'