Let’s imagine, molecular diagnostic report by Sanger sequencing shows a critical mutation in your patient based on which you will have to change the treatment regimen. Strangely, your patient’s clinical history doesn’t suggest you that s/he may have that particular mutation. Now you believe you should crosscheck this laboratory’s data. Should you be sending the sample to another laboratory? In that case, how will you explain your patient who is already burdened by the cost of test and subsequent cost of targeted therapy?
You can always crosscheck the diagnostic lab data right at your laptop. Sanger sequencing machine output comes as “electropherogram”. During DNA sequencing, each one of the A, T, C or G is labeled by a color dye. When a specific nucleotide is scanned by laser and detector within the capillary of Sanger sequencing machine, a peak of specific color is generated. Area under the peak (ie how high the peak is) is proportional to the signal strength. In other words, the more distinct and large the peak is, the surer you would be about the nucleotide it indicates. How can you read electropherogram? A very capable free software called SeqTrace can be downloaded from google code https://code.google.com/archive/p/seqtrace/ , right onto your laptop. Quick instructions for its usage can be found at its webpage. SeqTrace and its capabilities are published by its authors in Journal of Biomolecular Technology ( http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413935/ ) .
OK, now what? Now that you know how to analyze electropherogram, you need to know the rules of “base calling”. Base calling is the term used to describe the manual activity of seeing electropherogram and ascribing each peak, a nucleotide or otherwise. You can find rules of base calling, in guidelines of various organizations that regulate sequencing. One such document can be found at http://www.acgs.uk.com/media/774826/sequencingv2.pdf .
Now you ask your diagnostic lab to email you Sanger sequencing data as “.ab1”, “.scf”,”.ztr” files which can be opened in SeqTrace software. Voila!!! You are good to go.