Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-6-13

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Revision as of 17:50, 13 June 2017 by >Cjwei (Created page with "=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...")
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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

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'