DNA sequence traces are often used in cases where:
- We want to identify the source of the nucleic acid.
- We want to detect drug-resistant variants of human immune deficiency virus.
- We want to know which base is located at which position, especially where we might be able to diagnose a human disease or determine the best dose of a therapeutic drug.
In the future, these assays will likely rely more on automation. Currently, (at least outside of genome centers) many of these results are assessed by human technicians in clinical research labs, or DNA testing companies, who review these data by eye. (Yes, really!)
Today, you too, can be a data analyst and figure out what's happening with these traces.
These traces came from PCR products that were obtained from bacterial cultures by Sanger dideoxy sequencing. The bacterial cultures were isolated by JHU students in 2004. Some questions might have multiple answers.
- Which of these traces looks like it might have been generated from a mixed sample of DNA? (in this case, a mixed culture of bacteria.)
- Which of these traces probably came from a pure culture of bacteria?
- Which of these traces appears to contain positions with polymorphic bases? (Polymorphic means more than one form)
- The Extra Credit Question: All of these traces came from independently-isolated bacteria and are not likely to be the same species. Even so, two of the traces appear to have very similar sequences. Why do you think this might be the case? You can use blastn to answer this, but be sure to adjust the parameters for short sequences.
And, if you want to download some of our data, you can see how to get it here.
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Hi Sandra,
1) The answer to this question is C. Here we can clearly see that there secondary peaks at many base locations which can interfere with accurate base calling.
2) Traces A and B come from pure culture.
3) Traces A and B. There are polymorphic locations in A(169,171 etc) as compared to B. Also the base call quality is high so these can be considered as good or true changes.
4) I havent done any BLAST analysis. but i guess from the trace picture its looks like the sequences A & B are related or share same function or role but have diverged over a period of time. the entire pairwise alignment is good except there a couple of bases in trace A at location 181 and 185 which have poor quality values (which may be sequencing errors)
Amit: you are right in that all three sequences are related. They were generated from different samples, but using same set of primers.
And you're right about which cultures are mixed and which are not and about the bases that differ between samples. But, you're not right about the polymorphisms. Only one of these samples contains positions with polymorphic bases.
Hi Sandra,
Trace A appears to contain positions with polymorphic bases. If we clone this PCR product and pick several colonies to be extracted and sequenced (as plasmid DNA), the mixed base position at 181 and 184-base of Trace A could show as 2 different bases in separate clones.
Angela: You are correct, although I think these sequences would be tough to clone.
Hi,
please,in my samples i can see the bases in reigon with back ground gry and also on back ground pink with program AB1 for sequencing what this mean? and can i depend on the bases in gry region or not
thanks
Are you viewing traces in FinchTV from iFinch or GSAE?
If so, the area with the pink shading is the area that matches the vector and the area in gray is the area with poor quality sequence that would be trimmed by other programs in the Geospiza system. So, if this is the case, the answer is no.