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=sci-Methyl Seq Barcode 1 Design v3; Barcode 2 Design v2=
=sci-Methyl Seq Barcode 1 Design v4; Barcode 2 Design v3=
==Background==
==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:
*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:
Line 6: Line 6:
**Create final PCR primers that will add the P5/P7 sequencing adapters to both ends of the DNA fragment
**Create final PCR primers that will add the P5/P7 sequencing adapters to both ends of the DNA fragment
==Overview==
==Overview==
*Below is a general overview of the experimental procedure to ligate Adpt1 and Adpt2 (same as <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-6-12> but with PCR added in)
*Below is a general overview of the experimental procedure to ligate Adpt1 and Adpt2 (same as <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-6-12> but with PCR added in).  '''NOTE: <span style="color:red">NoG</span> sequences are in <span style="color:red">red/H</span>, <span style="color:blue">NoC</span> sequences are in <span style="color:blue">blue/D</span>.'''
  <u>End Repair/dA-Tailing</u>
  <u>End Repair/dA-Tailing</u>
       5'  '''-----'''A 3'
       5'  '''-----'''A 3'
       3' A'''-----'''  5'
       3' A'''-----'''  5'
             |
             |
             V
             V
  <u>Add Adpt1_v3:</u>     5' /5Phos/-----TTDD[Barcode1]-----CC 3'
   
                   3'      T-----AA''DD''[''Barcode1'']----- 5'
Add Adpt1_v4:    5' /5Phos/<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3'
                   3'      <u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5'
             |
             |
             V
             V
  <u>Ligate Adpt1_v3 (using same optimized ligation protocol as before)</u>
       5'  -----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----CC 3'
  <u>Ligate Adpt1_v4 (using same optimized ligation protocol as before)</u>
       3' CC-----[Barcode1]DDTT-----|A'''-----'''|T-----AA''DD''[''Barcode1'']----- 5'
       5'  <span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3'
       3' <span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5'
             |
             |
             V
             V
  <u>Add Adpt2_v2:</u>    5' /5Phos/-----[''Barcode2'']'''''DDDDDDDD'''''----- 3'
                   3'      GG-----[Barcode2]'''DDDDDDDD'''----- 5'
  <u>Add Adpt2_v3:</u>    5' /5Phos/<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3'
                   3'      <span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5'
             |
             |
             V
             V
  <u>Ligate Adpt2_v2 (using same optimized ligation protocol as before)</u>
  <u>Ligate Adpt2_v3 (using same optimized ligation protocol as before)</u>
                                       Nick
                                       Nick
                                       V
                                       V
       5' -----'''DDDDDDDD'''[Barcode2]-----GG|-----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----CC|-----[''Barcode2'']'''''DDDDDDDD'''''----- 3'
       5' <span style="color:red">-----'''HHHHHHHH'''[Barcode2]-----GG</span>|<span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3'
       3' -----'''''DDDDDDDD'''''[''Barcode2'']-----|CC-----[Barcode1]DDTT-----|A'''-----'''|T-----AA''DD''[''Barcode1'']-----|GG-----[Barcode2]'''DDDDDDDD'''----- 5'
       3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span>|<span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5'
                                                                                                ^
                                                                                                    ^
                                                                                                Nick
                                                                                                    Nick
             |
             |
             V
             V
  <u>Denature/separate fragments (will break up DNA at nicks)</u>
  <u>Denature/separate fragments (will break up DNA at nicks)</u>
    5' -----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----CC|-----[''Barcode2'']'''''DDDDDDDD'''''----- 3'
                                    5' <span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3'
    3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5'
   
   
    3' -----'''''DDDDDDDD'''''[''Barcode2'']-----|CC-----[Barcode1]DDTT-----|A'''-----'''|T-----AA''DD''[''Barcode1'']----- 5'
             |
             |
             V
             V
  <u>Bisulfite conversion</u>
  <u>Bisulfite conversion</u> (C->U)
             |
             |
             V
             V
  <u>PCR, 1st cycle (Add P7 sequencing adapters)</u>
  <u>PCR (P7 will be added first followed by P5)</u>
   P5: 5' AATGATACGGCGACCACCGA 3'
   P5: 5' AATGATACGGCGACCACCGA 3'
   P7: 5' CAAGCAGAAGACGGCATACGAGAT 3'
   P7: 5' CAAGCAGAAGACGGCATACGAGAT 3'
    5' -----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----CC|-----[''Barcode2'']'''''DDDDDDDD'''''----- 3'
                                                                                      3' <-----
      5' <span style="color:blue">-----[''Barcode1'']''DD''AA-----</span><u>AUT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----UU</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''----- 3'</span>
                                                                                                \
        ---------------------------------------------------------------------------------- 3' <-----
                                                                                                P7 5'
        -----> 3'                                                                                  \
   5' P7
        /                                                                                            P7 5'
       \
  5' P5
         -----> 3'
     3' -----'''''DDDDDDDD'''''[''Barcode2'']-----|CC-----[Barcode1]DDTT-----|A'''-----'''|T-----AA''DD''[''Barcode1'']----- 5'
                                                                                                    P5
   5' P7                                                                                           /
       \                                                                                     <-----
         -----> 3' ----------------------------------------------------------------------------------
     3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">UU-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TUA</u><span style="color:blue">-----AA''DD''[''Barcode1'']----- 5' </span>
             |
             |
             V
             V
<u>PCR, 2nd cycle (Add P5 sequencing adapters)</u>
  5' P5
      \
      -----> 3'
    3' -----[Barcode1]DDTT-----A|'''-----'''T|-----AA''DD''[''Barcode1'']-----GG|-----[Barcode2]'''DDDDDDDD'''-----P7 5'
   
   
    5' P7-----'''DDDDDDDD'''[Barcode2]-----|GG-----[''Barcode1'']''DD''AA-----|T'''-----'''|A-----TTDD[Barcode1]----- 3'
                                                                                        3' <-----
                                                                                                  \
                                                                                                  P5 5'
            |
            V
  <u>PCR Product</u>
  <u>PCR Product</u>
  5' P5-----[''Barcode1'']''DD''AA-----T|'''-----'''A|-----TTDD[Barcode1]-----CC|-----[''Barcode2'']'''''DDDDDDDD'''''-----P7 3'
  5' P5<span style="color:blue">-----[''Barcode1'']''DD''AA-----</span><u>AAT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----AA</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span>P7 3'
      ----->            ----->                                      ----->
      Index2 Primer      Read1 Primer                                Index2 Primer


==Barcode 1 Adapter Design (Adpt1_v4)==
==Potential Filler Sequences (noG's)==
*We want to further modify Adpt1 from Adpt1_v3 (which added the additional CC sticky end for Adpt2 ligation) by chainging 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.
*We want to gather a list of potential filler sequences we can use in adapter design that contain no G's.
*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' /5Phos/-----<u>ACA|GT</u>-----TTDD[Barcode1]-----CC 3'
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
*We want to ensure melting temperature of sequence is ~56C-60C (ideal temperature of 58C, which matches P5/P7 adapters) with a length of 18-30bp.  Consequently, we want to rerun the primergenerator.py script again to generate more potential sequences (see more information on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-3-13> and <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-4-28>).  Below are the new parameters we want to set:
*We want to ensure melting temperature of sequence is ~56C-60C (ideal temperature of 58C, which matches P5/P7 adapters) with a length of 18-30bp.  Consequently, we want to rerun the primergenerator.py script again to generate more potential sequences (see more information on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-3-13> and <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-4-28>).  Below are the new parameters we want to set:
**Set the temperature options to: tmoupt=58, tmmin=56, tmmax=60
**Set the temperature options to: tmoupt=58, tmmin=56, tmmax=60
**Use the noG variant of the script in order to form Filler 2 and Filler 3 (Adpt2)
**Use the noG variant of the script in order to form Filler 2 and Filler 3 (Adpt2)
**Set length parameter (-l, --plength) to 22 in order to compensate for the slightly increased melting temperature
**Set length parameter (-l, --plength) to 22 in order to compensate for the slightly increased melting
*Below are the potential Filler 1 sequences:
*Below are some potential noG sequences that may be used as filler sequences:
                         Tm (C)    Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
                         Tm(Py Script)    Tm(Oligo Analyzer)    Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
  '''<u>GCACACATAGCACGACGCGATT 56.7</u>'''
  CCCACTATCATCTACCCTCACC 56.7             56.2                  Pass
  TTGACTGCTGTGGAGTCGTTCG 56.7
ACTCCATCCCTCCACCCCTATC  58.6              59.9                  Pass
  GTACCTCGCCCGTTACCTTCGT 58.6       Warning: There are more than 3 self-annealing bases
  CCACTCCACCACTCCTCACCTA 58.6              60.1                  Pass
  GTTGGGCCGACACACTTGAGAA 56.7
  CCATTCTCCACTCCACCACACC 58.6             59.9                  Pass
  TGAGGCTCACTATGCGATCCTC 56.7       Warning: There are more than 3 self-annealing bases; There are more than 3 hairpin bases
TTCCCATCTCTACTCTCCTCCC  56.7                                    There are more than 3G's or C's in the last 5 bases
  AAGTGCGGACCCCCAATGCATC 58.6       Warning: There are more than 3 self-annealing bases
  <u>CCTCACCCCTCTTTCCATACAC 56.7             57.1                  Pass</u>
  GTGGGTCGACAAAGCATCCCCT 58.6       Warning; There are more than 3 Gs or Cs in the last 5 bases
  <u>CCACCCCATTAAACCCACCAAC 56.7             58.6                  Pass</u>
  GTGTCTGCCACTGCCTCTCTTT 56.7
CCCAACCAAAACATCCCCCTCC  58.6                                    There are more than 3G's or C's in the last 5 bases
  TAGCCGACTGGGAAGTCCCCAT 58.6       Warning: There are more than 3 self-annealing bases; There are more than 3 hairpin bases
  CCCTTTTCCCACCCTTCTCCCA 58.6             61.3                  Pass
  GGCCTACGACTACTGTCTGCGA 58.6      Warning: There are more than 3 self-annealing bases (Also contains the HpyCH4III cut site in sequence)
CTCACTTCTCTCACCTACTCCC  56.7                                    There are more than 3G's or C's in the last 5 bases
*Below are the potential Filler 2/3 (noG) sequences:
  AACCCCTCATTACAACCCCCCC 58.6                                     Contains runs of C's; There are more than 3G's or C's in the last 5 bases
                        Tm (C)    Primer Stats Notes
  CACCCTCCTTCTACCTAAACCC 56.7             56.6                  Pass
  CATCTCCACTACCCCCCAACCT 58.6       Warning: Contains run of C's
  TTACTTCCCCACCACCCACCCT 58.6             62.5                  Pass
  TTCACCTCACCTTACCACCCCC 58.6       Warning: Contains run of C's; There are more than 3 G's or C's in the last 5 bases
  <u>CCATTTCCTCACTCCCACCCAA 56.7              59.3                   Pass</u>
  '''<u>CAACCTCCACCTCACTCTCCTC 58.6</u>'''
  CACACTCCACCTCTTCCCCCTT 58.6                                     Contains runs of C's
  TCCATCTTCCCACCCATCTCCC 58.6       Warning: Contains run of C's; There are more than 3 G's or C's in the last 5 bases
  CCCCAAATCCTCCCCTTCTACC 58.6             59.2                  Pass
  CCCCACACTCCCCCAAAACCAA 58.6       Warning: Contains run of C's
TATCCTCCCCCATTCCTCCTCA  56.7                                    Contains runs of C's
  TCCCCCCCCTCATACTCACTTC 58.6       Warning: Contains run of C's
CTAACCCATCCCCCTTCCACTA  56.7                                    Contains runs of C's
  TCCCCCCCTCAACTCAACACTC 58.6       Warning: Contains run of C's
ACCCCCTACTCCACCCACATTT  56.7                                    Contains runs of C's
  TCCACACAACCCCCCAACCTCT 58.6       Warning: Contains run of C's
  TATCTCTCCCCCACCCTACCTA 56.7                                    Contains runs of C's
  '''<u>TCCCTCCCATTATCTCCACCCT 56.7</u>'''
  CCCCCACAATCACCACAACTCC 58.6                                     Contains runs of C's
  ATTACATCCACCCCCCTCACTC 56.7      Warning: Contains run of C's
ACCACCATCTCCATCCTCCACC  58.6                                    There are more than 3G's or C's in the last 5 bases
*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)
  ACCCAACCACTCTCACCCCTCT 58.6             62.2                  Pass
                                      Filler2 (originally Filler 2 sequence was GTCCCTCCTACCCGGCGTTT, but need to change such that no G's)
  <u>CCACCTCTTTCCCTCCTCAACC 58.6             59.4                  Pass</u>
                                        |                               Filler1 (Originally was omitted in Adpt1 because was extraneous, but need to add back in order to form second strand)
TACCCCCTCTCCACACACATAC  56.7                                    Contains runs of C's
                                        |                                 |
  CCTTCCTCCTCCACATCTTCCC 58.6             59                    Pass
                                        V                                 V
TCCCCTATCACCCCCAACTTCT  56.7                                    Contains runs of C's
  Adpt1_v4:       5' /5Phos/GTTCG<u>ACA|GT'''CAACCTCCACCTCACTCTCCTCTT'''</u>AG[AGGTG]<u>'''GCACACATAGCACGACGCGATT'''</u>CC 3'
  CCCTACCCCTCCACCTCAATCA 58.6             60.3                  Pass
                            ^
CCCTCTCCACACCATTCTTACC  56.7              57.1                  Pass
                            |
CCACTTAAATCCTCCCCCCACA  56.7                                    Contains runs of C's
                          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' AATCGCGTCGTGCTATGTGTGC 3' (Complementary to Filler 1 in order to do second strand synthesis)
==Barcode 1 Adapter Design (Adpt1_v4)==
*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:
  <u>'''Order the following oligos:'''</u>
  Adpt1_v4:          5' -----<u>ACA|GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3'
Adpt1_v4_primer:                                3' <span style="color:blue">-----</span> 5'
   
  <u>'''Perform second strand synthesis:'''</u>
5' -----<u>ACA|GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3'
3' -----<u>TG|TCA</u><span style="color:blue">-----AADD[''Barcode1'']<----</span> 5'
<u>'''HpyCH4III RE digestion'''</u>
5'  <u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3'
3' <u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']<----</span> 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' TCACC<u>ACA|GT</u><span style="color:red">CCTCACCCCTCTTTCCATACAC'''TTAC'''[ACCTC]CCACCCCATTAAACCCACCAAC'''CC'''</span> 3' (NOTE: Must order Adpt1_v4 at 100nm because it is >60bp)
Adpt1_v4_primer:    5' <span style="color:blue">GTTGGTGGGTTTAATGGGGTGG</span> 3'
===Adpt1_v4 Prep===
*We want to use a similar protocol as <http://www.nature.com/nprot/journal/v9/n11/box/nprot.2014.170_BX1.html> to prep the oligos.  However, since this is only a test round for now, we want to use a smaller amount of oligos as input.  Below is the workflow we will be using:
Anneal oligos
        |
        V
Klenow exo- fill-in
        |
        V
Zymo purification
        |
        V
HpyCH4III digestion
        |
        V
  Zymo purification
 
==Barcode 2 Adapter Design (Adpt2_v3)==
*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:
<u>'''Order the following oligos:'''</u>
Adpt2_v3_primer:     5' /5Phos/<span style="color:blue">-----</span> 3'
Adpt2_v3:            3'      <span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5
<u>'''Perform second strand synthesis:'''</u>
5' /5Phos/<span style="color:blue">---->[''Barcode2'']''DDDDDDDD''-----</span> 3'
3'      <span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5
*Consequently, we want to order the following adapters:
Adpt2_v3_primer:    5' /5Phos/<span style="color:blue">GGTAAAGGAGTGAGGGTGGGTT</span> 3'
  Adpt2_v3:           5' <span style="color:red">CCAACTCCTCCCTTTCTCCACC'''CCTCTCCA'''[CCTATC]AACCCACCCTCACTCCTTTACC</span><u>GG</u> 3' (NOTE: We can still order 25nm amount from IDT since oligo is exaclty 60bp long)

Latest revision as of 19:25, 19 June 2017

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)