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==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: <span style="color:red">NoG</span> sequences are in <span style="color:red">red/H</span>, <span style="color:blue">NoC</span> sequences are in <span style="color:blue">blue/D</span>.''' <u>End Repair/dA-Tailing</u> 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' -----<u>ACA|GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN|GTG</u>----- 3' 3' <----- 5' | Second strand synthesis V 5' -----<u>ACA|GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN|GTG</u>----- 3' 3' -----<u>TG|TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span><u>GTG|NNNCAC</u>----- 5' | Cut by HpyCH4III V 5' /5Phos/<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN|GTG</u>----- 3' 3' <u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span><u>GTG|NNNCAC</u>----- 5' | Cut by DraIII V 5' /5Phos/<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN</u> 3' 3' <u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span><u>GTG</u>/5Phos/ 5' <u>Ligate Adpt1_v5 (using same optimized ligation protocol as before)</u> 5' <u>GTG</u><span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN</u> 3' 3' <u>NNNCAC</u><span style="color:red">-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span><u>GTG</u> 5' | V Adpt2_v4: (second strand synthesis; cut by DraIII) <span style="color:blue">3' / / /</span> 5' -----<u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- 3' ----- <span style="color:blue">\ \ \ 5'</span> | Second strand synthesis V <span style="color:blue">3' / / /</span> 5' -----<u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- 3' -----<u>GTG|''NNN''CAC</u><span style="color:red">-----[''Barcode2'']HHHHHHHH</span>----- <span style="color:blue">\ \ \ 5'</span> | Cut by DraIII V <span style="color:blue">3' / / /</span> 5' /5Phos/<u>GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- 3' <u>''NNN''CAC</u><span style="color:red">-----[''Barcode2'']HHHHHHHH</span>----- <span style="color:blue">\ \ \ 5'</span> <u>Ligate Adpt2_v4 (using same optimized ligation protocol as before)</u> <span style="color:blue">5' 3' \ / \ / \ /</span> -----<span style="color:red">HHHHHHHH[''Barcode2'']-----</span><u>CAC''NNN''|GTG</u><span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- -----<span style="color:blue">DDDDDDDD[Barcode2]-----</span><u>GTG|NNNCAC</u><span style="color:red">-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span><u>GTG|''NNN''CAC</u><span style="color:red">-----[''Barcode2'']HHHHHHHH</span>----- <span style="color:blue">/ \ / \ / \ 3' 5'</span> | V <u>Bisulfite conversion</u> (C->U) | V <u>PCR (P7 will be added first followed by P5)</u> P5: 5' AATGATACGGCGACCACCGA 3' P7: 5' CAAGCAGAAGACGGCATACGAGAT 3' 5' 3' P5 P7 <span style="color:blue">\ / \ / \ /</span> -----<span style="color:red">HHHHHHHH[''Barcode2'']-----</span><u>CAC''NNN''|GTG</u><span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- -----<span style="color:blue">DDDDDDDD[Barcode2]-----</span><u>GTG|NNNCAC</u><span style="color:red">-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span><u>GTG|''NNN''CAC</u><span style="color:red">-----[''Barcode2'']HHHHHHHH</span>----- <span style="color:blue">/ \ / \ / \ P7 P5</span> 3' 5' | V <u>Sequencing</u> (based on HiSeq 2500 Dual-Indexed on Single-Read Flow Cell [[File:cw_20170705_Indexed_Sequencing_Overview.pdf.gz]]) P5 P7 <span style="color:blue">\ / \ / \ /</span> -----<span style="color:red">HHHHHHHH[''Barcode2'']-----</span><u>CAC''NNN''|GTG</u><span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- -----> -----> Read1 Index1 (i7) -----> Index2 (i5) <u>Sequencing</u> (based on HiSeq 4000/MiSeq Dual-Indexed on Paired-Read Flow Cell) P5 P7 <span style="color:blue">\ / \ / \ /</span> -----<span style="color:red">HHHHHHHH[''Barcode2'']-----</span><u>CAC''NNN''|GTG</u><span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- -----> -----> (1) Read1 (2) Index1 (i7) <----- <----- (3) Index2 (i5) (4) Read2 <u>Sequencing</u> (based on HiSeq 2500 Dual-Indexed on Paired-Read Flow Cell) P5 P7 <span style="color:blue">\ / \ / \ /</span> -----<span style="color:red">HHHHHHHH[''Barcode2'']-----</span><u>CAC''NNN''|GTG</u><span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----</span><u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- -----xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx> -----> -----> (3) Index2 (i5) (Grafted p5 w/ chemistry-only cycles) (1) Read1 (2) Index1 (i7) <----- (4) Read2
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