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==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
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