Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-6-12

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Revision as of 14:57, 12 June 2017 by >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...")
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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 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'