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

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     3'                                                                                                                                              5'
     3'                                                                                                                                              5'


==Potential Filler Sequences (noG's)==
==Potential Filler Sequences==
*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 redesign the potential filler sequences to be used for the adapters. The purpose of the filler sequences are two fold: (1) they will be used as sequencing primer binding sites on the Illumina flowcell, and (2) the filler sequences at the end of Adpt2 will be used as primer sites for adding P5/P7 sequences.
*We want to gather a list of potential filler sequences we can use in adapter design that contain no G's.
*In order to satisfy the first purpose of the filler sequences, we want to ensure that they exhibit similar properties as standard Illumina sequencing primer sites.  This means that they must closely match the following parameters. This is based on <http://nextgen.mgh.harvard.edu/CustomPrimer.html> and [[File:cw_20170705_Illumina_Adapter_Sequences.pdf]]
*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:
Tm = 66C (recommended to use IDT oligoAnalyzer to calculate Tm using Neighbor Joining Method)
**Set the temperature options to: tmoupt=58, tmmin=56, tmmax=60
Length = 33bp
**Use the noG variant of the script in order to form Filler 2 and Filler 3 (Adpt2)
GC = 52%
**Set length parameter (-l, --plength) to 22 in order to compensate for the slightly increased melting
*We can also use truncated versions of these filler sequences as primer binding sites for adding the P5/P7 sequences during PCR (see the above Illumina Adapter Sequences document under Nextera adapters)
*Below are some potential noG sequences that may be used as filler sequences:
*In order to design these adapters, I'll be using a new custom script to generate random oligos ([[File:cw_20170705_primerGenerator.v2.txt]]).  The script will generate random sequences using the given parameters:
                        Tm(Py Script)    Tm(Oligo Analyzer)    Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
Length = 33bp
  CCCACTATCATCTACCCTCACC  56.7              56.2                  Pass
Min Tm = 65C
  ACTCCATCCCTCCACCCCTATC  58.6              59.9                  Pass
Max Tm = 70C
  CCACTCCACCACTCCTCACCTA  58.6              60.1                   Pass
GC content = 45-55%
  CCATTCTCCACTCCACCACACC  58.6              59.9                  Pass
No single base runs of C's or G's >3
  TTCCCATCTCTACTCTCCTCCC  56.7                                    There are more than 3G's or C's in the last 5 bases
No more than 3 C's or G's at either ends of the filler sequence
<u>CCTCACCCCTCTTTCCATACAC  56.7              57.1                  Pass</u>
*Below is the output of the script using the above parameters (<u>'''noC'''</u>):
  CCACCCCATTAAACCCACCAAC  56.7              58.6                  Pass
Seq                                    revComp(Seq)                           GC      Tm       IDT Tm   Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
  CCCAACCAAAACATCCCCCTCC  58.6                                    There are more than 3G's or C's in the last 5 bases
  GGTTGGTTGTGGTGGGTGATTGTGGTAGGGAAG      CTTCCCTACCACAATCACCCACCACAACCAACC      0.55    65.15    66        Pass
CCCTTTTCCCACCCTTCTCCCA  58.6              61.3                  Pass
  TGGTGGGATGGGTGAAGGTTGTTGGGTGTGATT      AATCACACCCAACAACCTTCACCCATCCCACCA      0.52    65.90    67        Pass
CTCACTTCTCTCACCTACTCCC  56.7                                    There are more than 3G's or C's in the last 5 bases
  GATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA      TCCCTCACTACCCATCACCTTCACCACCACATC      0.55    65.07    66.1     Pass
AACCCCTCATTACAACCCCCCC  58.6                                    Contains runs of C's; There are more than 3G's or C's in the last 5 bases
  TGGGAAGGGAAGGTGGAGTGGAAGTTGGAGAGT      ACTCTCCAACTTCCACTCCACCTTCCCTTCCCA      0.55    65.94    67.2      Pass
CACCCTCCTTCTACCTAAACCC  56.7              56.6                  Pass
  ATGTTGTGTGGAGGGTGTGGGTGGGATGAGAAA      TTTCTCATCCCACCCACACCCTCCACACAACAT      0.52    65.54    66.6      Pass
TTACTTCCCCACCACCCACCCT  58.6              62.5                  Pass
  GTAGGTGTTGGGTTTGGAAGGTGAGGTGGTGGT      ACCACCACCTCACCTTCCAAACCCAACACCTAC      0.55    65.85    66.9      Pass
  <u>CCATTTCCTCACTCCCACCCAA  56.7              59.3                  Pass</u>
  ATGGGAATGGGAGGGTGGGTGAGGTGGTAATGA      TCATTACCACCTCACCCACCCTCCCATTCCCAT      0.55    66.11    67.5      Pass
  CACACTCCACCTCTTCCCCCTT 58.6                                    Contains runs of C's
*Below is the output of the script for ACGT fragment in Adpt1 (will be digested away using DraIII)For this filler sequence, we want to decrease the melting temperature and length of the oligo to a normal PCR primer as follows (based on IDT primer design <https://www.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2013/10/21/designing-pcr-primers-and-probes>:
  CCCCAAATCCTCCCCTTCTACC  58.6              59.2                  Pass
  Tm = 60-64C
  TATCCTCCCCCATTCCTCCTCA 56.7                                    Contains runs of C's
  Length = 22
CTAACCCATCCCCCTTCCACTA  56.7                                    Contains runs of C's
  Seq                    revComp(Seq)            GC      Tm      IDT Tm    Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
  ACCCCCTACTCCACCCACATTT  56.7                                    Contains runs of C's
  TTACTGCCGTTGCGAACGCGAA TTCGCGTTCGCAACGGCAGTAA  0.55    60.24    62        Pass
  TATCTCTCCCCCACCCTACCTA 56.7                                    Contains runs of C's
  TGTGCGTTTGTGGCGCAACAAC GTTGTTGCGCCACAAACGCACA 0.55    60.12    61.9      More than 3 self-annealing/hairpin bases
CCCCCACAATCACCACAACTCC  58.6                                    Contains runs of C's
  TGACCACGTGCGTTCGTTGCAA TTGCAACGAACGCACGTGGTCA  0.55    60.36    62.3      More than 3 self-annealing bases
ACCACCATCTCCATCCTCCACC  58.6                                    There are more than 3G's or C's in the last 5 bases
  TGTGCGATGCGGCAATGACCAA  TTGGTCATTGCCGCATCGCACA 0.55    60.24    62.5      More than 50% of the bases are self annealing
  ACCCAACCACTCTCACCCCTCT 58.6              62.2                  Pass
  TGCTATGCTGCGCGGAATGCAA TTGCATTCCGCGCAGCATAGCA 0.55    60.37    62.5      More than 3 self-annealing bases
  <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 19:53, 5 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

  • We want to redesign the potential filler sequences to be used for the adapters. The purpose of the filler sequences are two fold: (1) they will be used as sequencing primer binding sites on the Illumina flowcell, and (2) the filler sequences at the end of Adpt2 will be used as primer sites for adding P5/P7 sequences.
  • In order to satisfy the first purpose of the filler sequences, we want to ensure that they exhibit similar properties as standard Illumina sequencing primer sites. This means that they must closely match the following parameters. This is based on <http://nextgen.mgh.harvard.edu/CustomPrimer.html> and File:Cw 20170705 Illumina Adapter Sequences.pdf
Tm = 66C (recommended to use IDT oligoAnalyzer to calculate Tm using Neighbor Joining Method)
Length = 33bp
GC = 52%
  • We can also use truncated versions of these filler sequences as primer binding sites for adding the P5/P7 sequences during PCR (see the above Illumina Adapter Sequences document under Nextera adapters)
  • In order to design these adapters, I'll be using a new custom script to generate random oligos (File:Cw 20170705 primerGenerator.v2.txt). The script will generate random sequences using the given parameters:
Length = 33bp
Min Tm = 65C
Max Tm = 70C
GC content = 45-55%
No single base runs of C's or G's >3
No more than 3 C's or G's at either ends of the filler sequence
  • Below is the output of the script using the above parameters (noC):
Seq                                     revComp(Seq)                            GC      Tm       IDT Tm    Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
GGTTGGTTGTGGTGGGTGATTGTGGTAGGGAAG       CTTCCCTACCACAATCACCCACCACAACCAACC       0.55    65.15    66        Pass
TGGTGGGATGGGTGAAGGTTGTTGGGTGTGATT       AATCACACCCAACAACCTTCACCCATCCCACCA       0.52    65.90    67        Pass
GATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA       TCCCTCACTACCCATCACCTTCACCACCACATC       0.55    65.07    66.1      Pass
TGGGAAGGGAAGGTGGAGTGGAAGTTGGAGAGT       ACTCTCCAACTTCCACTCCACCTTCCCTTCCCA       0.55    65.94    67.2      Pass
ATGTTGTGTGGAGGGTGTGGGTGGGATGAGAAA       TTTCTCATCCCACCCACACCCTCCACACAACAT       0.52    65.54    66.6      Pass
GTAGGTGTTGGGTTTGGAAGGTGAGGTGGTGGT       ACCACCACCTCACCTTCCAAACCCAACACCTAC       0.55    65.85    66.9      Pass
ATGGGAATGGGAGGGTGGGTGAGGTGGTAATGA       TCATTACCACCTCACCCACCCTCCCATTCCCAT       0.55    66.11    67.5      Pass
Tm = 60-64C
Length = 22
Seq                     revComp(Seq)            GC      Tm       IDT Tm    Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
TTACTGCCGTTGCGAACGCGAA  TTCGCGTTCGCAACGGCAGTAA  0.55    60.24    62        Pass
TGTGCGTTTGTGGCGCAACAAC  GTTGTTGCGCCACAAACGCACA  0.55    60.12    61.9      More than 3 self-annealing/hairpin bases
TGACCACGTGCGTTCGTTGCAA  TTGCAACGAACGCACGTGGTCA  0.55    60.36    62.3      More than 3 self-annealing bases
TGTGCGATGCGGCAATGACCAA  TTGGTCATTGCCGCATCGCACA  0.55    60.24    62.5      More than 50% of the bases are self annealing
TGCTATGCTGCGCGGAATGCAA  TTGCATTCCGCGCAGCATAGCA  0.55    60.37    62.5      More than 3 self-annealing bases