Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-7-3: Difference between revisions

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     P7                                                                                                                                              P5</span>
     P7                                                                                                                                              P5</span>
     3'                                                                                                                                              5'
     3'                                                                                                                                              5'
==Potential Filler Sequences (noG's)==
*This is same as <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-6-13#Potential_Filler_Sequences_.28noG.27s.29>
*We want to gather a list of potential filler sequences we can use in adapter design that contain no G's.
*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
**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
*Below are some potential noG sequences that may be used as filler sequences:
                        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
<u>CCTCACCCCTCTTTCCATACAC  56.7              57.1                  Pass</u>
<u>CCACCCCATTAAACCCACCAAC  56.7              58.6                  Pass</u>
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
<u>CCATTTCCTCACTCCCACCCAA  56.7              59.3                  Pass</u>
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
<u>CCACCTCTTTCCCTCCTCAACC  58.6              59.4                  Pass</u>
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

Revision as of 23:47, 3 July 2017

sci-Methyl Seq Barcode 1 Design v5; Barcode 2 Design v4

Background

  • We want to make to major changes to the adapter designs:
    • Adpt2 Y-Adapter Design:
      • We want to redesign the adapter sequences in order to avoid having to use a single primer PCR reaction, which seems to be giving us some trouble. In addition, we would be unable to perform PCR after bisulfite conversion to add appropriate sequencing adapters since the ends of the fragments with Adp2 would contain the same sequence. Instead, we would need to use commercial kits to perform library construction (such as Accel-NGS Methyl-Seq <https://swiftbiosci.com/products/accel-ngs-methyl-seq-dna-library-kit/>). This could prove problematic as further optimization/cost would be added in order to use these commercial kits.
      • Instead, we want to redesign Adpt2 to be Y-adapters with unique sequences on both strands that serve as primer binding sites. The general design would be as follows:
                                      3'
                                     /
                                    /
                                   /
         5'  -----[Barcode2]UMI----
                           3' <----
                                   \
                                    \
                                     \
                                      5'
    • Use 3-base sticky end for Adpt1/Adpt2 ligation:
      • Previously, I've tried a CC/GG sticky end, which seems to still allow Adpt2 to be annealing directly onto the original template fragment non-specifically. Consequently, instead, we will try using a three-base sticky end to help with increasing specificity of Adpt1/Adpt2 ligation.
      • To achieve this, we will use a restriction enzyme to cut the ends of Adpt1/Adpt2 in order to create the 3-base sticky end (use DraIII-HF from NEB).
Note: NoG sequences in red/H, NoC sequences in blue/D
HpyCH4III: ACN|GT
           TG|NCA
DraIII: CACNNN|GTG
        GTG|NNNCAC
Adpt1: (cut by HpyCH4III and DraIII)
    5' /5Phos/GT-----TTHH[Barcode1]-----CACNNN 3'
    3'       TCA-----AADD[Barcode1]-----GTG    5'
Adpt2: (cut by DraIII)
                                                3'
                                               /
                                              /
                                             /
    5' /5Phos/GTG-----[Barcode2]DDDDDDDD-----
    3'     NNNCAC-----[Barcode2]HHHHHHHH-----
                                             \
                                              \
                                               \
                                                5'

Overview

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

           |
           V
Adpt1_v5: (second strand synthesis; cut by HpyCH4III and DraIII)
    5' -----ACA|GT-----TTHH[Barcode1]-----CACNNN|GTG----- 3'
                                 3' <-----GTG|NNNCAC----- 5'
                          | Second strand synthesis
                          V
    5' -----ACA|GT-----TTHH[Barcode1]-----CACNNN|GTG----- 3'
    3' -----TG|TCA-----AADD[Barcode1]-----GTG|NNNCAC----- 5'
                          | Cut by HpyCH4III
                          V
    5' /5Phos/GT-----TTHH[Barcode1]-----CACNNN|GTG----- 3'
    3'       TCA-----AADD[Barcode1]-----GTG|NNNCAC----- 5'
                          | Cut by DraIII
                          V
    5' /5Phos/GT-----TTHH[Barcode1]-----CACNNN 3'
    3'       TCA-----AADD[Barcode1]-----GTG    5'
Ligate Adpt1_v5 (using same optimized ligation protocol as before)
     5'    GTG-----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CACNNN 3'
     3' NNNCAC-----[Barcode1]HHTT-----TG|A-----|TCA-----AADD[Barcode1]-----GTG    5'
           |
           V
Adpt2_v4: (second strand synthesis; cut by DraIII)
                                                     3'
                                                    /
                                                   /
                                                  /
    5' -----CACNNN|GTG-----[Barcode2]DDDDDDDD-----
    3'                                       -----
                                                  \
                                                   \
                                                    \
                                                     5'
                          | Second strand synthesis
                          V
                                                     3'
                                                    /
                                                   /
                                                  /
    5' -----CACNNN|GTG-----[Barcode2]DDDDDDDD-----
    3' -----GTG|NNNCAC-----[Barcode2]HHHHHHHH-----
                                                  \
                                                   \
                                                    \
                                                     5'
                          | Cut by DraIII
                          V
                                            3'
                                           /
                                          /
                                         /
    5'    GTG-----[Barcode2]DDDDDDDD-----
    3' NNNCAC-----[Barcode2]HHHHHHHH-----
                                         \
                                          \
                                           \
                                            5'
Ligate Adpt2_v4 (using same optimized ligation protocol as before)
    5'                                                                                                                                              3'
     \                                                                                                                                             /
      \                                                                                                                                           /
       \                                                                                                                                         /
        -----HHHHHHHH[Barcode2]-----CACNNN|GTG-----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CACNNN|GTG-----[Barcode2]DDDDDDDD-----
        -----DDDDDDDD[Barcode2]-----GTG|NNNCAC-----[Barcode1]HHTT-----TG|A-----|TCA-----AADD[Barcode1]-----GTG|NNNCAC-----[Barcode2]HHHHHHHH-----
       /                                                                                                                                         \
      /                                                                                                                                           \
     /                                                                                                                                             \
    3'                                                                                                                                              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'                                                                                                                                              3'
    P5                                                                                                                                              P7
     \                                                                                                                                             /
      \                                                                                                                                           /
       \                                                                                                                                         /
        -----HHHHHHHH[Barcode2]-----CACNNN|GTG-----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CACNNN|GTG-----[Barcode2]DDDDDDDD-----
        -----DDDDDDDD[Barcode2]-----GTG|NNNCAC-----[Barcode1]HHTT-----TG|A-----|TCA-----AADD[Barcode1]-----GTG|NNNCAC-----[Barcode2]HHHHHHHH-----
       /                                                                                                                                         \
      /                                                                                                                                           \
     /                                                                                                                                             \
    P7                                                                                                                                              P5
    3'                                                                                                                                              5'

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