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=sci-Methyl Seq Barcode 1 Design v4; Barcode 2 Design v3= ==Background== *Just yesterday, we designed new sci-Methyl Seq Adapter 1 and Adapter 2 sequences and ordered them to test whether we could ligate on Adapter 2 instead of using the current annealing method. However, looking further into the protocol, it would be prudent to begin designing Adapter 1 such that it will be appropriate for bisulfite conversion and PCR. In order to do this, we need to do the following: **Adjust Filler 2 such that it does not contain any G's, which would result in C's in the final sequence that may be bisulfite converted (making PCR inefficient/impossible as primers need a consistent binding site for adding the P5/P7 adapter regions) **Begin testing the formation of dsDNA Adpt1/Adpt2. This would require using a method similar to the HpyCH4III digestion outlined here <http://www.nature.com/nprot/journal/v9/n11/box/nprot.2014.170_BX1.html> to create the T-tailed Adpt1. dsDNA Adpt2 can be created just by using polymerization/end-repair. **Create final PCR primers that will add the P5/P7 sequencing adapters to both ends of the DNA fragment ==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> 5' '''-----'''A 3' 3' A'''-----''' 5' | V Add Adpt1_v4: 5' /5Phos/<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' 3' <u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5' | V <u>Ligate Adpt1_v4 (using same optimized ligation protocol as before)</u> 5' <span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' 3' <span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5' | V <u>Add Adpt2_v3:</u> 5' /5Phos/<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' 3' <span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5' | V <u>Ligate Adpt2_v3 (using same optimized ligation protocol as before)</u> Nick V 5' <span style="color:red">-----'''HHHHHHHH'''[Barcode2]-----GG</span>|<span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' 3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span>|<span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5' ^ Nick | V <u>Denature/separate fragments (will break up DNA at nicks)</u> 5' <span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' 3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5' | 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' <span style="color:blue">-----[''Barcode1'']''DD''AA-----</span><u>AUT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----UU</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''----- 3'</span> ---------------------------------------------------------------------------------- 3' <----- -----> 3' \ / P7 5' 5' P5 P5 5' P7 / \ <----- -----> 3' ---------------------------------------------------------------------------------- 3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">UU-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TUA</u><span style="color:blue">-----AA''DD''[''Barcode1'']----- 5' </span> | V <u>PCR Product</u> 5' P5<span style="color:blue">-----[''Barcode1'']''DD''AA-----</span><u>AAT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----AA</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span>P7 3' -----> -----> -----> Index2 Primer Read1 Primer Index2 Primer ==Potential Filler Sequences (noG's)== *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 <u>CCTCACCCCTCTTTCCATACAC 56.7 57.1 Pass</u> <u>CCACCCCATTAAACCCACCAAC 56.7 58.6 Pass</u> 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 <u>CCATTTCCTCACTCCCACCCAA 56.7 59.3 Pass</u> 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 <u>CCACCTCTTTCCCTCCTCAACC 58.6 59.4 Pass</u> 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 ==Barcode 1 Adapter Design (Adpt1_v4)== *We want to modify Adapter 1 by keeping the same T-tail and CC-tail, but adjusting the filler sequences to correctly either have noC's or noG's. In addition, we want to be able to test whether we can adequately form dsDNA Adpt1_v4 by second strand synthesis and HpyCH4III restriction enzyme digestion. The steps for dsDNA Adpt1_v4 formation are outlined below: <u>'''Order the following oligos:'''</u> Adpt1_v4: 5' -----<u>ACA|GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' Adpt1_v4_primer: 3' <span style="color:blue">-----</span> 5' <u>'''Perform second strand synthesis:'''</u> 5' -----<u>ACA|GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' 3' -----<u>TG|TCA</u><span style="color:blue">-----AADD[''Barcode1'']<----</span> 5' <u>'''HpyCH4III RE digestion'''</u> 5' <u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' 3' <u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']<----</span> 5' (NOTE: Because we needed to add the restriction enzyme cut site, we see that there is an extra CA prior to the T overhang, which shouldn't affect primers that we design, but there is now a C we must account for. I added the additional CA sequence above) *Consequently, we want to order the following adapters: Adpt1_v4: 5' TCACC<u>ACA|GT</u><span style="color:red">CCTCACCCCTCTTTCCATACAC'''TTAC'''[ACCTC]CCACCCCATTAAACCCACCAAC'''CC'''</span> 3' (NOTE: Must order Adpt1_v4 at 100nm because it is >60bp) Adpt1_v4_primer: 5' <span style="color:blue">GTTGGTGGGTTTAATGGGGTGG</span> 3' ===Adpt1_v4 Prep=== *We want to use a similar protocol as <http://www.nature.com/nprot/journal/v9/n11/box/nprot.2014.170_BX1.html> to prep the oligos. However, since this is only a test round for now, we want to use a smaller amount of oligos as input. Below is the workflow we will be using: Anneal oligos | V Klenow exo- fill-in | V Zymo purification | V HpyCH4III digestion | V Zymo purification ==Barcode 2 Adapter Design (Adpt2_v3)== *We next want to modify Adapter 2 such that we can likewise form the dsDNA Adapter 2 using second strand synthesis. In addition, we must follow the same noC's/noG's restrictions on filler sequences as outlined above. The steps for dsDNA Adpt2_v3 formation are outlined below: <u>'''Order the following oligos:'''</u> Adpt2_v3_primer: 5' /5Phos/<span style="color:blue">-----</span> 3' Adpt2_v3: 3' <span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5 <u>'''Perform second strand synthesis:'''</u> 5' /5Phos/<span style="color:blue">---->[''Barcode2'']''DDDDDDDD''-----</span> 3' 3' <span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5 *Consequently, we want to order the following adapters: Adpt2_v3_primer: 5' /5Phos/<span style="color:blue">GGTAAAGGAGTGAGGGTGGGTT</span> 3' Adpt2_v3: 5' <span style="color:red">CCAACTCCTCCCTTTCTCCACC'''CCTCTCCA'''[CCTATC]AACCCACCCTCACTCCTTTACC</span><u>GG</u> 3' (NOTE: We can still order 25nm amount from IDT since oligo is exaclty 60bp long)
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