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==Barcode 2 Adapter Design== *We will derive Filler 3 using a similar method we used to derive Filler 2 using Dan's script (primergenerator.py). See <https://docs.google.com/document/d/1f6fUYy26eaqqJKdxxTBFgeVucB0uOuHPdfUKYiLDF9U/edit?usp=sharing> **Consequently, we want Filler 3 to have a Tm of ~58.8C-61.4C with a length of 20bp **'''However, unlike Filler 2, we want to make sure that Filler 3 does not contain any G's. Otherwise, the copied sequence when doing second strand synthesis would contain C's, which could potentially be converted during bisulfite conversion **Below is a table of potential Filler 3 sequences with the one we decided to use bolded (we want to also evaluate whether these sequences passed the self-annealing and hairpin formation tests from <http://www.bioinformatics.org/sms2/pcr_primer_stats.html>) CCCCTCCTCCCCTTTTCACC 57.9 pass (high GC content) <u>'''CCTCTCCTTACCTCCTCTCC'''</u> 55.9 pass AACCATCCCCCCCCTTCCTT 55.9 pass (warning, contains runs of C's) TACCTCCACCTCTCCCCCAC 57.9 pass (warning, contains runs of C's; high GC content; GC clamp warning) CCTCTCCACCCCATCCTATC 55.9 pass CCAAACCCCCCTTCTACCCT 55.9 pass (warning, contains runs of C's) ACTACCCCTACTCTCCCCCC 57.9 pass (warning, contains runs of C's; high GC content; GC clamp warning) ACTCTACCAACCCCCCCCAC 57.9 pass (warning, contains runs of C's; high GC content; GC clamp warning) TACCCCCCCCCCCTCCAAAA 57.9 pass (warning, contains runs of C's; high GC content) CCCCCCCCCACAAAATCTTC 55.9 pass (warning, contains runs of C's) *We will choose a random 8-base UMI and Barcode 2 to start with (make sure that these don't have any G's in them because they will be copied as C's in bisulfite conversion): UMI = CCTCTCCA Barcode 2 = CCTATC *The following is the final version of the test of Adapter 2: 5' CCTCTCCTTACCTCCTCTCC'''CCTCTCCA'''[CCTATC]AAACGCCGGGTAGGAGGG<u>TG</u> 3' ^ | Two base mismatch so not exactly reverse complementary to the original Filler 2 sequence
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