This is perhaps more of a philosophical question - I am wondering for Sanger sequencing of a PCR fragment specifically - but is it necessary to do biological replicates for clean Sanger sequencing data?
For example, I recently sequenced an exon of a gene in 2 cell lines - for each of the two cell lines my template DNA came from a pool of 100,000 cells. I PCRed up the fragment and then sequenced it. Is it necessary to repeat this experiment? After all, the template DNA came from many thousands of cells. While the experiment is easy to repeat I'm curious to know whether it's necessary or expected.
I don't think is necessary or expected. You'll have the same result. If your DNA comes from cloning it, it's usually a good practice to have several clones sequenced to confirm the correctness of the sequence.
If you sequenced the PCR amplicon directly without intermediate cloning steps, there is no need to repeat the experiment. For PCR to introduce a mutation detectable upon direct sequencing (i.e. no cloning involded), a misincorporation would have to occurr at exactly the same position in a significant fraction of the amplicons during a PCR cycle. That is not likely to occur during the first cycle, and even less so as the PCR reaction progresses and the number of molecules that would comprise a 'significant' fraction increases exponentially.
OTOH, repeating a couple of PCR reactions and sending them to a sequencing facility costs next to nothing and will disarm the objections of many people.
By the way, in your case I'd worry more about validating the identity of the cell lines.
Thanks for the thoughts!
Yes - no cloning was involved. Glad to hear that it's not a necessary practice.
We've done the experiment a couple of times (optimizing the PCR to clean up the sequence data) and have gotten the same result both times though the most recent time was much more convincing (likely higher DNA quality post-PCR).
It's interesting you bring up IDing cell lines as that is essentially what we are doing. Both cell lines are directly from reputable repositories but one of them has had identification issues in the past to the point that ATCC does not sell them any longer (we did not obtain the cells from ATCC). However, the cell line of questionable identity has a single base pair insertion in one exon of our gene of interest reported in the literature - our in-house cell line appears to have that same mutation - so it's likely the cell line we think it is (and we're primarily interested in studying the nature of this mutation anyway).
No need for additional sequencing multiple times. I would however recommend sequencing the PCR fragments from both ends if you want to make sure that the sequence is correct (hence have each bp sequenced twice from either end). This increases the confidence much more than using the same primer twice. In Sanger sequencing you see that not all 'peaks' are equally high, sometimes some are quite low and misinterpreted in the analysis. Sometimes a single nt or repeat sequence is missed completely due to a skipping of the polymerase, you sometimes observe this when there is a stretch of the same nucleotide (e.g. 6 or more). It doesn't happen often, but I've seen it happen. Sequencing from the other end increases the confidence as the secondary structure of the template will be different and 'rare' skipping events are basically eliminated.
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