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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: 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> ==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 ==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 (<u>'''noC'''</u>): Seq revComp(Seq) GC Tm IDT Tm Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html) <u>GGTTGGTTGTGGTGGGTGATTGTGGTAGGGAAG CTTCCCTACCACAATCACCCACCACAACCAACC 0.55 65.15 66 Pass</u> (Adpt1_v5) <u>TGGTGGGATGGGTGAAGGTTGTTGGGTGTGATT AATCACACCCAACAACCTTCACCCATCCCACCA 0.52 65.90 67 Pass</u> (Adpt1_v5) <u>GATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA TCCCTCACTACCCATCACCTTCACCACCACATC 0.55 65.07 66.1 Pass</u> (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 ATGGGAATGGGAGGGTGGGTGAGGTGGT<u>AATGA</u> 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) <u>GAGGTGGAGGGAGAAGTGAAAGTTG CAACTTTCACTTCTCCCTCCACCTC 0.52 57.00 59.1</u> (Adpt2_v4 Barcode Seq Y-Adpt) <u>GGTTTTGGGTGGGATTAGTGATTGG CCAATCACTAATCCCACCCAAAACC 0.48 56.06 58.1</u> (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 *Below is the output of the script for ACGT fragment in Adpt1/Adpt2. This fragment will be similar to a normal PCR primer as it will only be used for creating the dsDNA extension and not for sequencing. 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>: Tm = 60-64C Length = 22 Seq revComp(Seq) GC Tm IDT Tm Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html) <u>TTACTGCCGTTGCGAACGCGAA TTCGCGTTCGCAACGGCAGTAA 0.55 60.24 62 Pass</u> (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 <u>TGAAACGCGGCAACACGGGAAA TTTCCCGTGTTGCCGCGTTTCA 0.55 60.37 62.5 Pass</u> (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)== *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' -----<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> 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) <u>Adpt1_v5 Barcode Seq</u> (This is top strand containing Barcode1 Sequence; filler sequences are all from revComp column) 5' AATGA<u>ACAGT</u><span style="color:red">CTTCCCTACCACAATCACCCACCACAACCAACC</span>TTAC[ACCTC]<span style="color:red">AATCACACCCAACAACCTTCACCCATCCCACCA</span><u>CACCAGGTG</u>TTCGCGTTCGCAACGGCAGTAA 3' <u>Adpt1_v5 Universal Primer</u> (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/<u>GT</u><span style="color:red">CTTCCCTACCACAATCACCCACCACAACCAACC</span>TTAC[ACCTC]<span style="color:red">AATCACACCCAACAACCTTCACCCATCCCACCA</span><u>CACCAG</u> 3' Adpt1_v5_comp: 5' /5Phos/<u>GTG</u>TGGTGGGATGGGTGAAGGTTGTTGGGTGTGATTGAGGTGTAAGGTTGGTTGTGGTGGGTGATTGTGGTAGGGAAG<u>ACT</u> 3' ==Barcode 2 Adapter Design (Adpt2_v4)== *As described above, the following is how we would eventually create dsDNA Adpt2_v4: 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> *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]) <u>Adpt2_v4 Barcode Seq</u> (This is top strand containing Barcode2 Sequence) 5' AATGA<u>CACCAGGTG</u><span style="color:blue">GATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA[GATAGG]TGGAGAGG</span>TGAAACGCGGCAACACGGGAAA<span style="color:blue">''GAGGTGGAGGGAGAAGTGAAAGTTG''</span> 3' (italicized portion is part of Y-tail) <u>Adpt2_v4 Universal Primer</u> (Primer to start second strand synthesis and complete Y-adapter formation) 5' <span style="color:blue">''GGTTTTGGGTGGGATTAGTGATTGG''</span>TTTCCCGTGTTGCCGCGTTTCA 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/<u>GTG</u><span style="color:blue">GATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA[GATAGG]TGGAGAGG</span>TGAAACGCGGCAACACGGGAAA<span style="color:blue">''GAGGTGGAGGGAGAAGTGAAAGTTG''</span> 3' (italicized portion is part of Y-tail) Adpt2_v4_comp: 5' <span style="color:blue">''GGTTTTGGGTGGGATTAGTGATTGG''</span>TTTCCCGTGTTGCCGCGTTTCACCTCTCCACCTATCTCCCTCACTACCCATCACCTTCACCACCACATC<u>CACCTG</u> 3' ==P5/P7 PCR Primers== *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: <u>Adpt2_v4 P5 Primer</u> 5' AATGATACGGCGACCACCGA<span style="color:blue">''GGTTTTGGGTGGGATTAGTGATTGG''</span> 3' <u>Adpt2_v4 P7 Primer</u> 5' CAAGCAGAAGACGGCATACGAGAT<span style="color:red">''CAACTTTCACTTCTCCCTCCACCTC''</span> 3'
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