Sanger sequencing Result: Difference between forward and reverse DNA strands
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4 months ago

From sanger sequencing result, the forward strand sequence is slightly different ( mismatch) from reverse one. Which strand should be considered for detection of mutation with reference sequence?

Forward sequence partial Chromatogram

Reverse sequence partial chromatogram

When they aligned together, two different mismatch were found in position no 227 G> and 245 C> in forward strand seq(1st image) in position no 290 C> and 308 T> in reverse strand sequence (2nd image, reverse complement)


Forwar-seq

reverse-seq

SNP sequence • 675 views
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Are you sure? That's not normal.

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Here, sequence chromatogram is added. Please look into it. When they aligned together, two different mismatch were found in position no 227 G> and 245 C> in forward strand seq(1st image) in position no 290 C> and 308 T> in reverse strand sequence (2nd image, reverse complement)

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Please tell me this is not patient data you are putting up on the web!

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Actually, that was thalassemia patients data.

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Did the patient consent to that?

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4 months ago

The second sequence is the reverse complement sequence of the first one. So the variant 227G>A is 309 C>T in the second. And 245C>G is 290 G>C in the second. Looks all good to me.

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4 months ago
h.mon 32k

Did you look at the chromatograms? Are there any visible artifacts in the region where the difference is located? Did you check the quality? Is the difference in a region of low quality? Differences are common in such cases, but in general it is easy to tell apart the correct base call from the wrong one.

Otherwise, you need to provide more information. You could post snippets of the chromatograms, for example.

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