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
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:
- Adpt2 Y-Adapter Design:
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).
- Use 3-base sticky end for Adpt1/Adpt2 ligation:
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
- 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: NoG sequences are in red/H, NoC sequences are in blue/D.
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)
- 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 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