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

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sci-Methyl Seq Barcode 1 Design v4; Barcode 2 Design v3[edit]

Background[edit]

  • 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[edit]

End Repair/dA-Tailing
     5'  -----A 3'
     3' A-----  5'

           |
           V

Add Adpt1_v4:     5' /5Phos/GT-----TTHH[Barcode1]-----CC 3'
                  3'       TCA-----AADD[Barcode1]----- 5'

           |
           V

Ligate Adpt1_v4 (using same optimized ligation protocol as before)
     5'   -----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CC 3'
     3' CC-----[Barcode1]HHTT-----TG|A-----|TCA-----AADD[Barcode1]----- 5'

           |
           V

Add Adpt2_v3:     5' /5Phos/-----[Barcode2]DDDDDDDD----- 3'
                  3'      GG-----[Barcode2]HHHHHHHH----- 5'

           |
           V

Ligate Adpt2_v3 (using same optimized ligation protocol as before)
                                     Nick
                                      V
     5' -----HHHHHHHH[Barcode2]-----GG|-----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3'
     3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]HHTT-----TG|A-----|TCA-----AADD[Barcode1]-----|GG-----[Barcode2]HHHHHHHH----- 5'
                                                                                                    ^
                                                                                                   Nick

           |
           V

Denature/separate fragments (will break up DNA at nicks)
                                   5' -----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3'

    3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]HHTT-----TG|A-----|TCA-----AADD[Barcode1]----- 5'

           |
           V

Bisulfite conversion (C->U)

           |
           V

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

     5' -----[Barcode1]DDAA-----AUT|-----A|GT-----TTHH[Barcode1]-----UU|-----[Barcode2]DDDDDDDD----- 3'
        ---------------------------------------------------------------------------------- 3' <-----
        -----> 3'                                                                                   \
       /                                                                                             P7 5'
  5' P5

                                                                                                    P5
  5' P7                                                                                            /
      \                                                                                      <-----
       -----> 3' ----------------------------------------------------------------------------------
    3' -----DDDDDDDD[Barcode2]-----|UU-----[Barcode1]HHTT-----TG|A-----|TUA-----AADD[Barcode1]----- 5' 

           |
           V

PCR Product
5' P5-----[Barcode1]DDAA-----AAT|-----A|GT-----TTHH[Barcode1]-----AA|-----[Barcode2]DDDDDDDD-----P7 3'
     ----->             ----->                                       ----->
     Index2 Primer      Read1 Primer                                 Index2 Primer

Potential Filler Sequences (noG's)[edit]

                        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)[edit]

  • We want to modify Adapter 1 by keeping the same T-tail and CC-tail, but adjusting the filler sequences to correctly either have noC's or noG's. In addition, we want to be able to test whether we can adequately form dsDNA Adpt1_v4 by second strand synthesis and HpyCH4III restriction enzyme digestion. The steps for dsDNA Adpt1_v4 formation are outlined below:
Order the following oligos:
Adpt1_v4:          5' -----ACA|GT-----TTHH[Barcode1]-----CC 3'
Adpt1_v4_primer:                                 3' ----- 5'

Perform second strand synthesis:
5' -----ACA|GT-----TTHH[Barcode1]-----CC 3'
3' -----TG|TCA-----AADD[Barcode1]<---- 5'

HpyCH4III RE digestion
5'  GT-----TTHH[Barcode1]-----CC 3'
3' TCA-----AADD[Barcode1]<---- 5' (NOTE: Because we needed to add the restriction enzyme cut site, we see that there is an extra CA prior to the T overhang, which shouldn't affect primers that we design, but there is now a C we must account for.  I added the additional CA sequence above)
  • Consequently, we want to order the following adapters:
Adpt1_v4:            5' TCACCACA|GTCCTCACCCCTCTTTCCATACACTTAC[ACCTC]CCACCCCATTAAACCCACCAACCC 3' (NOTE: Must order Adpt1_v4 at 100nm because it is >60bp)
Adpt1_v4_primer:     5' GTTGGTGGGTTTAATGGGGTGG 3'

Adpt1_v4 Prep[edit]

Anneal oligos
       |
       V
Klenow exo- fill-in
       |
       V
Zymo purification
       |
       V
HpyCH4III digestion
       |
       V
Zymo purification

Barcode 2 Adapter Design (Adpt2_v3)[edit]

  • We next want to modify Adapter 2 such that we can likewise form the dsDNA Adapter 2 using second strand synthesis. In addition, we must follow the same noC's/noG's restrictions on filler sequences as outlined above. The steps for dsDNA Adpt2_v3 formation are outlined below:
Order the following oligos:
Adpt2_v3_primer:     5' /5Phos/----- 3'
Adpt2_v3:            3'      GG-----[Barcode2]HHHHHHHH----- 5

Perform second strand synthesis:
5' /5Phos/---->[Barcode2]DDDDDDDD----- 3'
3'      GG-----[Barcode2]HHHHHHHH----- 5
  • Consequently, we want to order the following adapters:
Adpt2_v3_primer:     5' /5Phos/GGTAAAGGAGTGAGGGTGGGTT 3'
Adpt2_v3:            5' CCAACTCCTCCCTTTCTCCACCCCTCTCCA[CCTATC]AACCCACCCTCACTCCTTTACCGG 3' (NOTE: We can still order 25nm amount from IDT since oligo is exaclty 60bp long)