I have a small doubt in sequencing. I had sequenced 16s rRna region of my bacterium in two labs. Both of them had used different sets of primers for the same sample and same region and inferred the two as two different organism. How is this possible? Is it because of the variation in the primers?
NOTE: UNIVERSAL PRIMERS WERE USED BY BOTH TH E LABS. Thank you
Swetha,
1) do you have pure culture of your bacteria?
2) how far were two obtained sequences located on taxonomic tree?
If your culture was a mix of 2 or more bacteria - the result is expected.
Alex
Swetha,
what I would do if I were in such position:
I would make PCR1 using one set of primers, I would make second PCR using the seocnd set of primers, put both products in real time qPCR using sybergreen and determine their Tm profile.
If you get multiple peaks, then you have a mixed sample.
You could also make DNA restriction profile of the two productss and check whether the fragments make a simple and clear profile and the lengths add up.
Or, you can clone the product 1, clone the product2, make some 25 restriction profiles of each and see whether they are the same. If the yre not, then you have a mixed culture, or contamination during sequencing.
Best,
Blaz
Dear Swetha
I agree with Alex. If you are using soil DNA extract, than it is expected to be a mixture of hundreds to thousands of different bacteria. However, using universal primers the dominant ones will be amplified, and also using two different primers may have amplification bias towards two different bacterial communities. So, you can either clone the PCR products and sequence as much colonies possible, or else go for NGS with the soil DNA extract. You will get your answers.
SD
I use 20F and 1540R (E.coli number system) for near full lenght PCR products of 1521bp, with respect to E. coli for any new isolated pure strain.
This not only amplify full DNA but also all nine hypervariable regions, V1 to V9 (Chakravorty S. 2007). Before going for cloning what you can actually do is find out how many hypervariable regions you can get for both the sequences even though your sequence might not be full lenght.
By using simple BLAST2, you can find out hypervariable region in your bacterial sequence by giving option subject as E. coli and Query as your sequence and find out as many as V1 to V9 regions.
For preliminary studies even if you are successful in getting V3 and V6 or any one of then, then Blast the one region of one of your sequenc in NCBI. Same you do for 2nd sequence. Now, compare V3 of both the sequence, compare V6 of both the sequence, and find out the differences.
For guidance, I am sending one paper from our lab, although my student added information from his thesis in the form of paper, but this details may be helpful before going for costly cloning and re-do kind of things.
Best wishes.
Dr. Devendra Lingojwar
Director ATG LAB
www.biotechtraininingproject.com
Article Comparative studies on hypervariable regions of 16S ribosoma...
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