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

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Revision as of 20:06, 14 June 2017 by >Cjwei (→‎Overview)
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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/-----TTHH[Barcode1]-----CC 3'
                  3'       T-----AADD[Barcode1]----- 5'

           |
           V

Ligate Adpt1_v3 (using same optimized ligation protocol as before)
     5'   -----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC 3'
     3' CC-----[Barcode1]HHTT-----|A-----|T-----AADD[Barcode1]----- 5'

           |
           V

Add Adpt2_v2:     5' /5Phos/-----[Barcode2]DDDDDDDD----- 3'
                  3'      GG-----[Barcode2]HHHHHHHH----- 5'

           |
           V

Ligate Adpt2_v2 (using same optimized ligation protocol as before)
                                     Nick
                                      V
     5' -----HHHHHHHH[Barcode2]-----GG|-----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3'
     3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]HHTT-----|A-----|T-----AADD[Barcode1]-----|GG-----[Barcode2]HHHHHHHH----- 5'
                                                                                                ^
                                                                                               Nick

           |
           V

Denature/separate fragments (will break up DNA at nicks)
                                   5' -----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3'

    3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]HHTT-----|A-----|T-----AADD[Barcode1]----- 5'

           |
           V

Bisulfite conversion

           |
           V

PCR (P7 will be added first followed by P5)
 P5: 5' AATGATACGGCGACCACCGA 3'
 P7: 5' CAAGCAGAAGACGGCATACGAGAT 3'

     5' -----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3'
        ------------------------------------------------------------------------------ 3' <-----
        -----> 3'                                                                               \
       /                                                                                         P7 5'
  5' P5

                                                                                                 P5
  5' P7                                                                                         /
      \                                                                                   <-----
       -----> 3' -------------------------------------------------------------------------------
    3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]HHTT-----|A-----|T-----AADD[Barcode1]----- 5' 

           |
           V

PCR Product
5' P5-----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD-----P7 3'
     ----->             ----->                                   ----->
     Index2 Primer      Read1 Primer                             Index2 Primer

Potential Filler Sequences (noG's)

                        Tm(Py Script)     Tm(Oligo Analyzer)     Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
CCCACTATCATCTACCCTCACC  56.7              56.2                   Pass
ACTCCATCCCTCCACCCCTATC  58.6              59.9                   Pass
CCACTCCACCACTCCTCACCTA  58.6              60.1                   Pass
CCATTCTCCACTCCACCACACC  58.6              59.9                   Pass
TTCCCATCTCTACTCTCCTCCC  56.7                                     There are more than 3G's or C's in the last 5 bases
CCTCACCCCTCTTTCCATACAC  56.7              57.1                   Pass
CCACCCCATTAAACCCACCAAC  56.7              58.6                   Pass
CCCAACCAAAACATCCCCCTCC  58.6                                     There are more than 3G's or C's in the last 5 bases
CCCTTTTCCCACCCTTCTCCCA  58.6              61.3                   Pass
CTCACTTCTCTCACCTACTCCC  56.7                                     There are more than 3G's or C's in the last 5 bases
AACCCCTCATTACAACCCCCCC  58.6                                     Contains runs of C's; There are more than 3G's or C's in the last 5 bases
CACCCTCCTTCTACCTAAACCC  56.7              56.6                   Pass
TTACTTCCCCACCACCCACCCT  58.6              62.5                   Pass
CCATTTCCTCACTCCCACCCAA  56.7              59.3                   Pass
CACACTCCACCTCTTCCCCCTT  58.6                                     Contains runs of C's
CCCCAAATCCTCCCCTTCTACC  58.6              59.2                   Pass
TATCCTCCCCCATTCCTCCTCA  56.7                                     Contains runs of C's
CTAACCCATCCCCCTTCCACTA  56.7                                     Contains runs of C's
ACCCCCTACTCCACCCACATTT  56.7                                     Contains runs of C's
TATCTCTCCCCCACCCTACCTA  56.7                                     Contains runs of C's
CCCCCACAATCACCACAACTCC  58.6                                     Contains runs of C's
ACCACCATCTCCATCCTCCACC  58.6                                     There are more than 3G's or C's in the last 5 bases
ACCCAACCACTCTCACCCCTCT  58.6              62.2                   Pass
CCACCTCTTTCCCTCCTCAACC  58.6              59.4                   Pass
TACCCCCTCTCCACACACATAC  56.7                                     Contains runs of C's
CCTTCCTCCTCCACATCTTCCC  58.6              59                     Pass
TCCCCTATCACCCCCAACTTCT  56.7                                     Contains runs of C's
CCCTACCCCTCCACCTCAATCA  58.6              60.3                   Pass
CCCTCTCCACACCATTCTTACC  56.7              57.1                   Pass
CCACTTAAATCCTCCCCCCACA  56.7                                     Contains runs of C's



Barcode 1 Adapter Design (Adpt1_v4)

  • We want to further modify Adpt1 from Adpt1_v3 (which added the additional CC sticky end for Adpt2 ligation) by changing the Filler 2 sequence such that it does not contain any G's. The reason for this is because we want the complementary sequence of Filler 2 (which would have C's) to be changed during bisulfite conversion.
  • Below is the general design that we want to achieve with Adapter 1 (including the HpyCH4III cut site at the end):
                                     Filler2 (originally Filler 2 sequence was GTCCCTCCTACCCGGCGTTT, but need to change such that no G's)
                                        |                 Filler1 (Originally was omitted in Adpt1 because was extraneous, but need to add back in order to form second strand)
                                        |                   |
                                        V                   V
Adpt1_v4:       5' -----ACA|GT-----TTDD[Barcode1]-----CC 3' (RE cut will leave behind the necessary 5Phos group)
Adpt1_v4_comp:  3'   ^                          <-----   5' (Complementary to Filler 1 in order to do second strand synthesis)
                     |
                 Add 5bp flanking region that is enough for RE cut
                        Tm (C)     Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
GCACACATAGCACGACGCGATT  56.7
TTGACTGCTGTGGAGTCGTTCG  56.7
GTACCTCGCCCGTTACCTTCGT  58.6       Warning: There are more than 3 self-annealing bases
GTTGGGCCGACACACTTGAGAA  56.7
TGAGGCTCACTATGCGATCCTC  56.7       Warning: There are more than 3 self-annealing bases; There are more than 3 hairpin bases
AAGTGCGGACCCCCAATGCATC  58.6       Warning: There are more than 3 self-annealing bases
GTGGGTCGACAAAGCATCCCCT  58.6       Warning; There are more than 3 Gs or Cs in the last 5 bases
GTGTCTGCCACTGCCTCTCTTT  56.7
TAGCCGACTGGGAAGTCCCCAT  58.6       Warning: There are more than 3 self-annealing bases; There are more than 3 hairpin bases
GGCCTACGACTACTGTCTGCGA  58.6       Warning: There are more than 3 self-annealing bases (Also contains the HpyCH4III cut site in sequence)
  • Below are the potential Filler 2/3 (noG) sequences:
                        Tm (C)     Primer Stats Notes
CATCTCCACTACCCCCCAACCT  58.6       Warning: Contains run of C's
TTCACCTCACCTTACCACCCCC  58.6       Warning: Contains run of C's; There are more than 3 G's or C's in the last 5 bases
CAACCTCCACCTCACTCTCCTC  58.6
TCCATCTTCCCACCCATCTCCC  58.6       Warning: Contains run of C's; There are more than 3 G's or C's in the last 5 bases
CCCCACACTCCCCCAAAACCAA  58.6       Warning: Contains run of C's
TCCCCCCCCTCATACTCACTTC  58.6       Warning: Contains run of C's
TCCCCCCCTCAACTCAACACTC  58.6       Warning: Contains run of C's
TCCACACAACCCCCCAACCTCT  58.6       Warning: Contains run of C's
TCCCTCCCATTATCTCCACCCT  56.7
ATTACATCCACCCCCCTCACTC  56.7       Warning: Contains run of C's
  • Consequently, using the above Filler 1/2 sequences, we can make the necessary Adapter 1 oligos as follows: (using a set Barcode 1 [AGGTG] and DD [AG] sequence)
                                     Filler2 (originally Filler 2 sequence was GTCCCTCCTACCCGGCGTTT, but need to change such that no G's)
                                        |                                Filler1 (Originally was omitted in Adpt1 because was extraneous, but need to add back in order to form second strand)
                                        |                                  |
                                        V                                  V
Adpt1_v4:       5' GTTCGACA|GTGCACACATAGCACGACGCGATTTTAG[AGGTG]CAACCTCCACCTCACTCTCCTCCC 3'
                     ^
                     |
                  Add 5bp flanking region that is enough for RE cut (just choose a random sequence that will be cut off anyways)
Adpt1_v4_comp:  5' GAGGAGAGTGAGGTGGAGGTTG 3' (Complementary to Filler 1 in order to do second strand synthesis)

Barcode 2 Adapter Design (Adpt2_v3)

  • We want to again further modify Adpt2_v2 (which was previously designed to use the ligation method) by using the following:
    • Change Filler 3 sequence such that it contains no G's (otherwise, the complement would contain C's that may be converted during bisulfite conversion. This would confound PCR off of the filler sequence)
    • Add a Filler 4 sequence that will be used to form the second strand to create dsDNA Adapter 2 sequences (we will use the above