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

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sci-Methyl Seq Barcode 1 Design v5; Barcode 2 Design v4[edit]

Background[edit]

  • 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[edit]

End Repair/dA-Tailing
        First CG of every read contains true methylation signal
         V
     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' <----- 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/5Phos/    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' /5Phos/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'
           |
           V

Sequencing (based on HiSeq 2500 Dual-Indexed on Single-Read Flow Cell File:Cw 20170705 Indexed Sequencing Overview.pdf.gz)
    P5                                                                                                                                              P7
     \                                                                                                                                             /
      \                                                                                                                                           /
       \                                                                                                                                         /
        -----HHHHHHHH[Barcode2]-----CACNNN|GTG-----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CACNNN|GTG-----[Barcode2]DDDDDDDD-----
                                                                 ----->                                              ----->
                                                                 Read1                                               Index1 (i7)
                                              ----->
                                              Index2 (i5)

Sequencing (based on HiSeq 4000/MiSeq Dual-Indexed on Paired-Read Flow Cell)
    P5                                                                                                                                              P7
     \                                                                                                                                             /
      \                                                                                                                                           /
       \                                                                                                                                         /
        -----HHHHHHHH[Barcode2]-----CACNNN|GTG-----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CACNNN|GTG-----[Barcode2]DDDDDDDD-----
                                                                 ----->                                              ----->
                                                                 (1) Read1                                           (2) Index1 (i7)
                                                                 <-----                                              <-----
                                                                 (3) Index2 (i5)                                     (4) Read2

Sequencing (based on HiSeq 2500 Dual-Indexed on Paired-Read Flow Cell)
    P5                                                                                                                                              P7
     \                                                                                                                                             /
      \                                                                                                                                           /
       \                                                                                                                                         /
        -----HHHHHHHH[Barcode2]-----CACNNN|GTG-----[Barcode1]DDAA-----ACT|-----A|GT-----TTHH[Barcode1]-----CACNNN|GTG-----[Barcode2]DDDDDDDD-----
        -----xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx>             ----->                                              ----->
        (3) Index2 (i5) (Grafted p5 w/ chemistry-only cycles)    (1) Read1                                           (2) Index1 (i7)
                                                                                                                     <-----
                                                                                                                     (4) Read2

Potential Filler Sequences[edit]

  • 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 (Adpt1_v5)
TGGTGGGATGGGTGAAGGTTGTTGGGTGTGATT       AATCACACCCAACAACCTTCACCCATCCCACCA       0.52    65.90    67        Pass (Adpt1_v5)
GATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA       TCCCTCACTACCCATCACCTTCACCACCACATC       0.55    65.07    66.1      Pass (Adpt2_v4 Barcode Seq)
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
ATGAATGTGTGTGGGAGGATGGTGTGGGAAGGG       CCCTTCCCACACCATCCTCCCACACACATTCAT       0.55    65.83    66.9      Pass
  • We also want to determine suitable Y-adapter ends that need to contain noC's and also have slightly different parameters than above to make them suitable PCR adapters:
    • Make sure that the melting temperature difference between the two Y-adapters are within 5C apart since these will be used for PCR
Tm = 55-65C (need to slightly decrease melting temperature and increase primer length in order to obtain good primers; only a few degrees lower Tm/few bases longer than recommended by IDT)
Length = 25
Seq                             revComp(Seq)                    GC      Tm       IDT Tm    Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html)
GAGGTGGAGGGAGAAGTGAAAGTTG       CAACTTTCACTTCTCCCTCCACCTC       0.52    57.00    59.1 (Adpt2_v4 Barcode Seq Y-Adpt)
GGTTTTGGGTGGGATTAGTGATTGG       CCAATCACTAATCCCACCCAAAACC       0.48    56.06    58.1 (Adpt2_v4 Universal Primer Y-Adpt)
TGGAATGGTTAAGGTGGGAGGAAAG       CTTTCCTCCCACCTTAACCATTCCA       0.48    56.41    58.7
AGGGTGAGAGGGATGTGTAGGGTAA       TTACCCTACACATCCCTCTCACCCT       0.52    57.88    60.6
TTGGTTAGAGGTGGTTGGAGAGTGG       CCACTCTCCAACCACCTCTAACCAA       0.52    57.87    60.2
TGAAGTAGAGGTAGGTGAGAGGGTA       TACCCTCTCACCTACCTCTACTTCA       0.48    55.14    57.7
AAGGAGAGGGTAATAGGGAGTTTGG       CCAAACTCCCTATTACCCTCTCCTT       0.48    55.22    57.7
GAAGGTGTGGGTTGTAGTTTGAAGG       CCTTCAAACTACAACCCACACCTTC       0.48    56.00    57.8
GGGATGTGTTGGTAGTAGGGATTGG       CCAATCCCTACTACCAACACATCCC       0.52    56.62    58.9
AGGGTTGGATTGTTGAGGTGGTGTT       AACACCACCTCAACAATCCAACCCT       0.48    58.49    60.7
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 (Adpt1_v5)
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
AACATCTGCGCGCGAAACCACT  AGTGGTTTCGCGCGCAGATGTT  0.55    60.24    62.3      Pass
TTGTTTCGGGAGCGGCAAAGCA  TGCTTTGCCGCTCCCGAAACAA  0.55    60.11    62.4      Pass
AACGCCCGTGCGGAAATTCGTT  AACGAATTTCCGCACGGGCGTT  0.55    60.49    62.5      More than 3 hairpin bases
ATTAGACGCGTGCTGCAGCGTT  AACGCTGCAGCACGCGTCTAAT  0.55    60.05    62        Pass
TGAAACGCGGCAACACGGGAAA  TTTCCCGTGTTGCCGCGTTTCA  0.55    60.37    62.5      Pass (Adpt2_v4)
TTGCAAGTGCGCGCGGAACTAA  TTAGTTCCGCGCGCACTTGCAA  0.55    60.49    62.4      More than 3 self-annealing bases
AGTGCGCGCGCCATTGAGTATT  AATACTCAATGGCGCGCGCACT  0.55    60.11    62.2      More than 3 self-annealing/hairpin bases
ACGACGCAAATTTGCGCGTTGC  GCAACGCGCAAATTTGCGTCGT  0.55    60.84    62.2      More than 3 self-annealing/hairpin bases

Barcode 1 Adapter Design (Adpt1_v5)[edit]

  • As described above, the following is how we would eventually create dsDNA Adpt1_v5:
Adpt1_v5: (second strand synthesis; cut by HpyCH4III and DraIII)
    5' -----ACA|GT-----TTHH[Barcode1]-----CACNNN|GTG----- 3'
                                                3' <----- 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'
  • Today, I'll be ordering two different oligos to perform second strand synthesis and restriction enzyme digestion: (using Barcode1 = ACCTC, HH = AC, NNN=CAG)
Adpt1_v5 Barcode Seq (This is top strand containing Barcode1 Sequence; filler sequences are all from revComp column)
5' AATGAACAGTCTTCCCTACCACAATCACCCACCACAACCAACCTTAC[ACCTC]AATCACACCCAACAACCTTCACCCATCCCACCACACCAGGTGTTCGCGTTCGCAACGGCAGTAA 3'
Adpt1_v5 Universal Primer (Primer to start second strand synthesis)
5' TTACTGCCGTTGCGAACGCGAA 3'
  • If we wanted to order the primers fully formed, then we would order the following: (remove the sequences that would otherwise be digested away)
Adpt1_v5: 5' /5Phos/GTCTTCCCTACCACAATCACCCACCACAACCAACCTTAC[ACCTC]AATCACACCCAACAACCTTCACCCATCCCACCACACCAG 3'
Adpt1_v5_comp: 5' /5Phos/GTGTGGTGGGATGGGTGAAGGTTGTTGGGTGTGATTGAGGTGTAAGGTTGGTTGTGGTGGGTGATTGTGGTAGGGAAGACT 3'

Barcode 2 Adapter Design (Adpt2_v4)[edit]

  • As described above, the following is how we would eventually create dsDNA Adpt2_v4:
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' /5Phos/GTG-----[Barcode2]DDDDDDDD-----
    3'     NNNCAC-----[Barcode2]HHHHHHHH-----
                                             \
                                              \
                                               \
                                                5'
  • Below are the two oligos I'll be ordering today to perform second strand synthesis and restriction enzyme digestion: (using Barcode2 = GATAGG, DDDDDDDD = TGGAGAGG, NNN=CAG [such that won't anneal to the A-tailed sticky end of the original template])
Adpt2_v4 Barcode Seq (This is top strand containing Barcode2 Sequence)
5' AATGACACCAGGTGGATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA[GATAGG]TGGAGAGGTGAAACGCGGCAACACGGGAAAGAGGTGGAGGGAGAAGTGAAAGTTG 3' (italicized portion is part of Y-tail)
Adpt2_v4 Universal Primer (Primer to start second strand synthesis and complete Y-adapter formation)
5' GGTTTTGGGTGGGATTAGTGATTGGTTTCCCGTGTTGCCGCGTTTCA 3' (italicized portion is part of Y-tail)
  • If we wanted to order the primers fully formed, then we would order the following: (remove the sequences that would otherwise be digested away)
Adpt2_v4: 5' /5Phos/GTGGATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA[GATAGG]TGGAGAGGTGAAACGCGGCAACACGGGAAAGAGGTGGAGGGAGAAGTGAAAGTTG 3' (italicized portion is part of Y-tail)
Adpt2_v4_comp: 5' GGTTTTGGGTGGGATTAGTGATTGGTTTCCCGTGTTGCCGCGTTTCACCTCTCCACCTATCTCCCTCACTACCCATCACCTTCACCACCACATCCACCTG 3'

P5/P7 PCR Primers[edit]

  • We want to design the P5/P7 PCR primers that will anneal to the Y-adapters and add on the appropriate sequencing adapters. Below are the sequences for P5/P7 regions required for sequencing (see File:Cw 20170706 Illumina Library Structure.pdf for information regarding the library structure)
P5: 5' AATGATACGGCGACCACCGA 3'
P7: 5' CAAGCAGAAGACGGCATACGAGAT 3'
  • Below are the two oligos I'll be ordering today to perform P5/P7 PCR of the final ligation products:
Adpt2_v4 P5 Primer
5' AATGATACGGCGACCACCGAGGTTTTGGGTGGGATTAGTGATTGG 3'
Adpt2_v4 P7 Primer
5' CAAGCAGAAGACGGCATACGAGATCAACTTTCACTTCTCCCTCCACCTC 3'