Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-6-13: Difference between revisions
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**Create final PCR primers that will add the P5/P7 sequencing adapters to both ends of the DNA fragment | **Create final PCR primers that will add the P5/P7 sequencing adapters to both ends of the DNA fragment | ||
==Overview== | ==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) | *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 <span style="color:red">red/H</span>, NoC sequences are in <span style="color:blue">blue/D</span>. | ||
<u>End Repair/dA-Tailing</u> | <u>End Repair/dA-Tailing</u> | ||
5' '''-----'''A 3' | 5' '''-----'''A 3' | ||
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V | V | ||
Add Adpt1_v3: 5' /5Phos/<span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' | |||
3' T----- | 3' <span style="color:blue">T-----AADD[''Barcode1'']-----</span> 5' | ||
| | | | ||
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<u>Ligate Adpt1_v3 (using same optimized ligation protocol as before)</u> | <u>Ligate Adpt1_v3 (using same optimized ligation protocol as before)</u> | ||
5' -----[''Barcode1''] | 5' <span style="color:blue">-----[''Barcode1'']DDAA-----T</span>|'''-----'''A|<span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' | ||
3' CC-----[Barcode1] | 3' <span style="color:red">CC-----[Barcode1]HHTT-----</span>|A'''-----'''|<span style="color:blue">T-----AADD[''Barcode1'']-----</span> 5' | ||
| | | | ||
V | V | ||
<u>Add Adpt2_v2:</u> 5' /5Phos/-----[''Barcode2'']'''''DDDDDDDD'''''----- 3' | <u>Add Adpt2_v2:</u> 5' /5Phos/<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' | ||
3' GG-----[Barcode2]''' | 3' <span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5' | ||
| | | | ||
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Nick | Nick | ||
V | V | ||
5' -----''' | 5' <span style="color:red">-----'''HHHHHHHH'''[Barcode2]-----GG</span>|<span style="color:blue">-----[''Barcode1'']DDAA-----T</span>|'''-----'''A|<span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' | ||
3' -----'''''DDDDDDDD'''''[''Barcode2'']-----|CC-----[Barcode1] | 3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">CC-----[Barcode1]HHTT-----</span>|A'''-----'''|<span style="color:blue">T-----AADD[''Barcode1'']-----</span>|<span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5' | ||
^ | ^ | ||
Nick | Nick | ||
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<u>Denature/separate fragments (will break up DNA at nicks)</u> | <u>Denature/separate fragments (will break up DNA at nicks)</u> | ||
5' <span style="color:blue">-----[''Barcode1'']DDAA-----T</span>|'''-----'''A|<span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' | |||
3' -----'''''DDDDDDDD'''''[''Barcode2'']-----|CC-----[Barcode1] | 3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">CC-----[Barcode1]HHTT-----</span>|A'''-----'''|<span style="color:blue">T-----AADD[''Barcode1'']-----</span> 5' | ||
| | | | ||
V | V | ||
<u>Bisulfite conversion</u> | <u>Bisulfite conversion</u> | ||
| | | | ||
V | V | ||
<u>PCR | <u>PCR (P7 will be added first followed by P5)</u> | ||
P5: 5' AATGATACGGCGACCACCGA 3' | P5: 5' AATGATACGGCGACCACCGA 3' | ||
P7: 5' CAAGCAGAAGACGGCATACGAGAT 3' | P7: 5' CAAGCAGAAGACGGCATACGAGAT 3' | ||
5' -----[''Barcode1'']''DD''AA-----T|'''-----'''A|----- | 5' <span style="color:blue">-----[''Barcode1'']''DD''AA-----T</span>|'''-----'''A|<span style="color:red">-----TTHH[Barcode1]-----CC</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">CC-----[Barcode1]HHTT-----</span>|A'''-----'''|<span style="color:blue"T-----AA''DD''[''Barcode1'']----- 5' </span> | |||
| | | | ||
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<u>PCR Product</u> | <u>PCR Product</u> | ||
5' P5-----[''Barcode1'']''DD''AA-----T|'''-----'''A|----- | 5' P5<span style="color:blue">-----[''Barcode1'']''DD''AA-----T</span>|'''-----'''A|<span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span>P7 3' | ||
==Potential Filler Sequences (noG's)== | ==Potential Filler Sequences (noG's)== |
Revision as of 19:49, 14 June 2017
sci-Methyl Seq Barcode 1 Design v3; Barcode 2 Design v2
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: NoG sequences are in red/H, NoC sequences are in blue/D.
End Repair/dA-Tailing 5' -----A 3' 3' A----- 5' | V Add Adpt1_v3: 5' /5Phos/-----TTHH[Barcode1]-----CC 3' 3' T-----AADD[Barcode1]----- 5' | V Ligate Adpt1_v3 (using same optimized ligation protocol as before) 5' -----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC 3' 3' CC-----[Barcode1]HHTT-----|A-----|T-----AADD[Barcode1]----- 5' | V Add Adpt2_v2: 5' /5Phos/-----[Barcode2]DDDDDDDD----- 3' 3' GG-----[Barcode2]HHHHHHHH----- 5' | V Ligate Adpt2_v2 (using same optimized ligation protocol as before) Nick V 5' -----HHHHHHHH[Barcode2]-----GG|-----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3' 3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]HHTT-----|A-----|T-----AADD[Barcode1]-----|GG-----[Barcode2]HHHHHHHH----- 5' ^ Nick | V Denature/separate fragments (will break up DNA at nicks) 5' -----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3' 3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]HHTT-----|A-----|T-----AADD[Barcode1]----- 5' | V Bisulfite conversion | V PCR (P7 will be added first followed by P5) P5: 5' AATGATACGGCGACCACCGA 3' P7: 5' CAAGCAGAAGACGGCATACGAGAT 3' 5' -----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD----- 3' ------------------------------------------------------------------------------ 3' <----- -----> 3' \ / P7 5' 5' P5 P5 5' P7 / \ <----- -----> 3' ------------------------------------------------------------------------------- 3' -----DDDDDDDD[Barcode2]-----|CC-----[Barcode1]HHTT-----|A-----|<span style="color:blue"T-----AADD[Barcode1]----- 5' | V PCR Product 5' P5-----[Barcode1]DDAA-----T|-----A|-----TTHH[Barcode1]-----CC|-----[Barcode2]DDDDDDDD-----P7 3'
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 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
Barcode 1 Adapter Design (Adpt1_v4)
- We want to further modify Adpt1 from Adpt1_v3 (which added the additional CC sticky end for Adpt2 ligation) by changing the Filler 2 sequence such that it does not contain any G's. The reason for this is because we want the complementary sequence of Filler 2 (which would have C's) to be changed during bisulfite conversion.
- Below is the general design that we want to achieve with Adapter 1 (including the HpyCH4III cut site at the end):
Filler2 (originally Filler 2 sequence was GTCCCTCCTACCCGGCGTTT, but need to change such that no G's) | Filler1 (Originally was omitted in Adpt1 because was extraneous, but need to add back in order to form second strand) | | V V Adpt1_v4: 5' -----ACA|GT-----TTDD[Barcode1]-----CC 3' (RE cut will leave behind the necessary 5Phos group) Adpt1_v4_comp: 3' ^ <----- 5' (Complementary to Filler 1 in order to do second strand synthesis) | Add 5bp flanking region that is enough for RE cut
- 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 temperature
- Below are the potential Filler 1/4 sequences:
Tm (C) Primer Stats Notes (http://www.bioinformatics.org/sms2/pcr_primer_stats.html) GCACACATAGCACGACGCGATT 56.7 TTGACTGCTGTGGAGTCGTTCG 56.7 GTACCTCGCCCGTTACCTTCGT 58.6 Warning: There are more than 3 self-annealing bases GTTGGGCCGACACACTTGAGAA 56.7 TGAGGCTCACTATGCGATCCTC 56.7 Warning: There are more than 3 self-annealing bases; There are more than 3 hairpin bases AAGTGCGGACCCCCAATGCATC 58.6 Warning: There are more than 3 self-annealing bases GTGGGTCGACAAAGCATCCCCT 58.6 Warning; There are more than 3 Gs or Cs in the last 5 bases GTGTCTGCCACTGCCTCTCTTT 56.7 TAGCCGACTGGGAAGTCCCCAT 58.6 Warning: There are more than 3 self-annealing bases; There are more than 3 hairpin bases GGCCTACGACTACTGTCTGCGA 58.6 Warning: There are more than 3 self-annealing bases (Also contains the HpyCH4III cut site in sequence)
- Below are the potential Filler 2/3 (noG) sequences:
Tm (C) Primer Stats Notes CATCTCCACTACCCCCCAACCT 58.6 Warning: Contains run of C's TTCACCTCACCTTACCACCCCC 58.6 Warning: Contains run of C's; There are more than 3 G's or C's in the last 5 bases CAACCTCCACCTCACTCTCCTC 58.6 TCCATCTTCCCACCCATCTCCC 58.6 Warning: Contains run of C's; There are more than 3 G's or C's in the last 5 bases CCCCACACTCCCCCAAAACCAA 58.6 Warning: Contains run of C's TCCCCCCCCTCATACTCACTTC 58.6 Warning: Contains run of C's TCCCCCCCTCAACTCAACACTC 58.6 Warning: Contains run of C's TCCACACAACCCCCCAACCTCT 58.6 Warning: Contains run of C's TCCCTCCCATTATCTCCACCCT 56.7 ATTACATCCACCCCCCTCACTC 56.7 Warning: Contains run of C's
- Consequently, using the above Filler 1/2 sequences, we can make the necessary Adapter 1 oligos as follows: (using a set Barcode 1 [AGGTG] and DD [AG] sequence)
Filler2 (originally Filler 2 sequence was GTCCCTCCTACCCGGCGTTT, but need to change such that no G's) | Filler1 (Originally was omitted in Adpt1 because was extraneous, but need to add back in order to form second strand) | | V V Adpt1_v4: 5' GTTCGACA|GTGCACACATAGCACGACGCGATTTTAG[AGGTG]CAACCTCCACCTCACTCTCCTCCC 3' ^ | Add 5bp flanking region that is enough for RE cut (just choose a random sequence that will be cut off anyways) Adpt1_v4_comp: 5' GAGGAGAGTGAGGTGGAGGTTG 3' (Complementary to Filler 1 in order to do second strand synthesis)
Barcode 2 Adapter Design (Adpt2_v3)
- We want to again further modify Adpt2_v2 (which was previously designed to use the ligation method) by using the following:
- Change Filler 3 sequence such that it contains no G's (otherwise, the complement would contain C's that may be converted during bisulfite conversion. This would confound PCR off of the filler sequence)
- Add a Filler 4 sequence that will be used to form the second strand to create dsDNA Adapter 2 sequences (we will use the above