Noi/NOTES/2011-9-13

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Sanger sequencing results of the amplicons amplified by randomly tagging primers with and without USER[edit]

  • Continued from 2011_09_07: [[1]]
  • 10 clones from the reaction with USER --> if there was no AmpFNU.Sol primer left over after USER digestion, we should observe every clone contains poly T sequences (TTTTTTT -> 7T) by reading with reverse primer, Syb_RP7.
    • 8 clones showed very clear poly T sequences (7T). One clone (NP-NA-U-01) showed only 6T and another clone (NP-NA-U-09) showed TTTCTTT instead of TTTTTTT. For the first one, I think that it should be the error of primer synthesis since the surrounded sequences were corrected (CTGAGAGTGTTTTTTGTGTAGA). For the second one, I am not sure if it derived from the AmpFNU.Sol primer or the artifact of sequencing since the DNA chromatogram showed a very clear C peak. However, based on the failure cause of the this sample, it said that there was a spectral pull-up that might be caused by too much DNA in the reaction. For me, I believed that it should derive from the AmpAU.Sol primer as the reason of spectral pull-up that T pulled up G, C then A.

File:NP-NA-U-01.png
File:NP-NA-U-09.png

  • 10 clones of positive control with no USER in the reaction --> 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)

2011_09_14
Sample NP-NA-U-09 which showed TTTCTTT sequences was re-sequenced again. This time, the peak still showed the same pattern as C peak looked very clean. However Dinh and I though that it's unlikely that the random primer (NU) will have these sequence. We still need to validate this by running in Illumina sequencing as Dr. Zhang suggested. File:NP-NA-U-09-repeated.png

  • To validate the number of AmpFNU.Sol primer left over after USER digestion, I plan to do the following experiment.
    • 1 reaction -> do the experiment as I previously did by using AmpFAU.Sol and AmpF6.3.Index primers in the first two cycles -> add 1ul of USER and AmpFNU.Sol -> Amplify with the short primers, Syb_FP5A and Syb_RP7 (the condition in details as previously used) -> SE sequencing by Illumina. After this, we can calculate the % of survival AmpFNU.sol primer after USER digestion, and decide if this number is acceptable to use in the future experiment.
    • To also get the data usable for the manuscript, I plan to set up another reaction but use AmpFNU.Sol instead of AmpFAU.Sol and another AmpF6.3Index primers. -> Add 1ul of USER -> Amplify with the short primers, Syb_FP5A and Syb_RP7.
  • The second reaction is just the optional choice since Dinh suggested that if the validation go well, we can try this experiment in the samples with the new probes synthesized from LC Sciences.


2012_06_18

Re-analyzed the results from Sanger sequencing by looking closer the chromatogram of each sample[edit]

Sample IDs Surrounding sequences
contain AGAGTG (7A or 7N) GTG
7A, AAAAAAA 7N Note: 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.

Chromatograms of the clones from 7A+7N+USER[edit]

Passed validation
File:NA-1U-01.png File:NA-1U-02.png File:NA-1U-03.png File:NA-1U-04.png File:NA-1U-05.png File:NA-1U-06.png File:NA-1U-07.png File:NA-1U-08.png File:NA-1U-10.png
Questionable
File:NA-1U-09.png

Chromatograms of the clones from 7A+7N-noUSER[edit]

PolyT (7T)
File:NA-1U-14.png File:NA-1U-15.png File:NA-1U-18.png
PolyN (7N)
File:NA-1U-11.png File:NA-1U-12 R.png File:NA-1U-13.png File:NA-1U-16.png File:NA-1U-17.png File:NA-1U-19.png File:NA-1U-20.png