Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-3-13

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sci-Methyl Seq Barcode 1 Design[edit]

Background[edit]

5’ [Phos]CCG----------TTDD[Barcode1]---------- 3’
            (Filler 1)              (Filler 2)

Barcode 1 Adapter Design[edit]

  • This is from the Google Doc here <https://docs.google.com/document/d/1f6fUYy26eaqqJKdxxTBFgeVucB0uOuHPdfUKYiLDF9U/edit?usp=sharing>
  • From the previous experimental design, we see that Filler 1 is somewhat extraneous (there is nothing that is intended to bind to Filler 1), while Filler 2 must have a high enough Tm in order for Adapter 2 annealing later on in the protocol.
    • To simplify design, we will just remove Filler 1 from the adapter sequence for now (may want to add in later if we want to evaluate bisulfite treatment efficiency)
  • Filler 2 Tm should be ~58.8C-61.4C with a length of 20bp. These parameters are based on the P5/P7 sequences that are appropriate for KAPA library amplification.
  • Using Dan's script to generate primers (primergenerator.py), we generated several random Filler 2 sequences we could potentially use. For the version 1 of the design, we'll be using the following Filler 2 sequence:
Sequence                 primerGenerator Tm     OligoAnalyzer Tm     Notes
GTCCCTCCTACCCGGCGTTT     57.9                   61.8 (58C)           Only 4-base self-dimer
  • We also want to choose an appropriate Barcode 1 (using a 3-letter alphabet [AGT] in order to avoid confounding results.
  • Consequently, the following is the final version of the test of Adapter 1:
5’ [Phos]CGTTAG[AGGTG]GTCCCTCCTACCCGGCGTTT 3’
           AADD[Barcode]------Filler 2----
    • We want to try making the Adapter 1 into a double stranded adapter, which may be necessary for the T4 ligation to work (need a bigger footprint than the 2-base overhang left by MspI digestion). Consequently, we want to anneal the following complementary sequence to Adapter 1:
5’ AAACGCCGGGTAGGAGGGACCACCTCTAA 3’
    • Initial tests without the dsDNA adapter resulted in no ligation.

PCR Primer Design[edit]

  • We want to perform a PCR reaction in order to amplify the ligated product to verify that Barcode 1 Adapter annealed correctly. In order to do this, we want to create the following primer:
Adpt1_PCR     5' AAACGCCGGGTAGGAGGGAC 3'
  • We only need one primer for PCR because there should be two identical dsDNA Adapter 1's annealed to both ends of the target fragment. The steps in PCR annealing is shown below:
5' AAACGCCGGGTAGGAGGGACCACCTCTAACG--------CGTTAGAGGTGGTCCCTCCTACCCGGCGTTT 3'
3' TTTGCGGCCCATCCTCCCTGGTGGAGATTGC--------GCAATCTCCACCAGGGAGGATGGGCCGCAAA 5'
                                 |
                                 V
Round 1 PCR:
 5' AAACGCCGGGTAGGAGGGAC--->
 3' TTTGCGGCCCATCCTCCCTGGTGGAGATTGC--------GCAATCTCCACCAGGGAGGATGGGCCGCAAA 5'
                                 |
                                 V
Round 2 PCR:
5' AAACGCCGGGTAGGAGGGACCACCTCTAACG--------CGTTAGAGGTGGTCCCTCCTACCCGGCGTTT 3'
                                                 <---CAGGGAGGATGGGCCGCAAA 5'