Hi, this is Dr. Harini, Manager with a genetic diagnostic lab. Recently we have done Sanger sequencing for PKD1 gene variant in proband and tested the fetus of the proband for the same. I need a clarification of the sequencing information as mentioned in the image attached. Can someone please clarify the same.
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No T insertion variant carried by the proband was detected at the corresponding PKD1 locus in the fetal sample.Originally posted by harini.k View Post
The weak A peak observed in the reverse sequencing chromatogram represents a sequencing artifact (trailing peak) rather than a true heterozygous T>A variant, as it does not exhibit the characteristic peak pattern of a heterozygous substitution and is not supported by the forward sequencing results. Therefore, this signal should not be interpreted as a genuine nucleotide substitution.
To further confirm the result, the following approaches are recommended:- Repeat reverse sequencing using a new PCR amplification and a fresh sequencing reaction.
- Design a short PCR amplicon (≤300 bp) flanking the insertion site.
- Perform cloning-based sequencing, if permitted by legal and ethical regulations.
- Alternatively, conduct next-generation sequencing (NGS) followed by manual inspection of the BAM files.
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