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==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: <span style="color:red">'''NoG'''</span> sequences in <span style="color:red">'''red/H'''</span>, <span style="color:blue">'''NoC'''</span> sequences in <span style="color:blue">'''blue/D'''</span> HpyCH4III: ACN|GT TG|NCA DraIII: CACNNN|GTG GTG|NNNCAC Adpt1: (cut by HpyCH4III and DraIII) 5' /5Phos/GT<span style="color:red">-----TTHH[Barcode1]-----</span>CACNNN 3' 3' TCA<span style="color:blue">-----AADD[Barcode1]-----</span>GTG 5' Adpt2: (cut by DraIII) <span style="color:blue">3' / / /</span> 5' /5Phos/GTG<span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- 3' NNNCAC<span style="color:red">-----[Barcode2]HHHHHHHH</span>----- <span style="color:blue">\ \ \ 5'</span>
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