Question: Mismatched frequencies of complementary bases after sequencing
gravatar for L. A. Liggett
3.5 years ago by
L. A. Liggett120
Broad Institute, Harvard Medical School, Boston Children's
L. A. Liggett120 wrote:

I am performing amplicon sequencing of the human genome, which just amplifies up regions of the genome and sequences them. Everything seems to work just fine, but I observe a phenomenon that I am unable to rationalize.

If I stratify variants by base change, (ie separately bin A -> T changes, A -> G changes, etc.) I find that complementary base change frequencies do not always match. For instance it would be expected that since DNA is double stranded, roughly every time an A -> G change is observed the complementary T -> C change should be observed.

This is almost always the case, but I do get repeatable strong mismatches in certain base mutation frequencies. For example something like 1000 observed A -> G variants but only 10 observed T -> C variants.

Biologically this does not make sense to me. Is there something that can account for this phenomenon?

sequencing next-gen genome • 771 views
ADD COMMENTlink modified 3.5 years ago by WouterDeCoster42k • written 3.5 years ago by L. A. Liggett120
gravatar for WouterDeCoster
3.5 years ago by
WouterDeCoster42k wrote:

Dependent on the structure of your nucleotide base:

Depending on whether your example was random or real, this is a partial answer.

In addition, most common mutation is methyl cytosine to T (oxidative deamination)

ADD COMMENTlink modified 3.5 years ago • written 3.5 years ago by WouterDeCoster42k

@WouterDeCoster this explains why C -> T and its complementary change of G -> A would be more prevalent in the data. But those two changes should be more or less equally observed in frequency. What I am seeing for some bases is the complementary bases not matching in frequency. So for this example it would be like G -> A is observed far more often than C -> T. Make sense?

ADD REPLYlink written 3.5 years ago by L. A. Liggett120
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