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== Re-analyzed the results from Sanger sequencing by looking closer the chromatogram of each sample == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Surrounding sequences<br> contain AGAGTG (7A or 7N) GTG''' | align="center" style="background:#f0f0f0;"|'''7A, AAAAAAA''' | align="center" style="background:#f0f0f0;"|'''7N''' | align="center" style="background:#f0f0f0;"|'''Note:''' | align="center" style="background:#f0f0f0;"|'''Pass validation''' |- | '''7A+7N+USER'''|||||||||| |- | NA-1U-01||yes||6A||||:6A instead of 7A||yes |- | NA-1U-02||yes||yes||||:Dirty PCR||yes |- | NA-1U-03||yes||yes||||:Homopolymeric or Repetitive Region||yes |- | NA-1U-04||yes||yes||||:Homopolymeric or Repetitive Region||yes |- | NA-1U-05||ATG instead of CTG||yes||||||yes |- | NA-1U-06||yes||yes||||||yes |- | NA-1U-07||yes||yes||||||yes |- | NA-1U-08||yes||yes||||:Peaks very low||yes |- | NA-1U-09||yes||TTTCTTT||||||?? |- | NA-1U-10||GTT instead of GTG||yes||||||yes |- | '''7A+7N-noUSER'''|||||||||| |- | NA-1U-11||yes||||AATACGT||||yes |- | NA-1U-12||yes||||CTTACTG||||yes |- | NA-1U-13||yes||||TAATCGT||||yes |- | NA-1U-14||yes||yes||||||yes |- | NA-1U-15||yes||yes||||:Homopolymeric or Repetitive Region||yes |- | NA-1U-16||yes||||CCATACC||||yes |- | NA-1U-17||yes||||GCAATTT||:Spectral Pull-up||yes |- | NA-1U-18||yes||yes||||||yes |- | NA-1U-19||yes||||AAATTCA||||yes |- | NA-1U-20||yes||||CAAATAG||||yes |} * '''Summary''' ** 10 clones from the reaction with USER (7A+7N+USER) *** 8 clones showed very clear poly T sequences (7T). *** 1 clone showed 6T instead of 7T *** 1 clone showed TTTCTTT instead of 7T ** 10 clones of positive control with no USER in the reaction '''(7A+7N-noUSER)''' --> we expected to see the mix of polyT (TTTTTTT -> 7T) and random sequences (NNNNNNN -> 7N) *** 3 clones with poly T (TTTTTTT -> 7T) *** 7 clones with random sequences (NNNNNNN -> 7N) ** From the sequences of 7N, most of the sequences showed random nucleotide and none of them showed poly T. Therefore it is most likely that one nucleotide deviated on the 7T sequences might be derived from primer synthesis. Will be discussed with Dr. Zhang and Athurva.
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