Is it possible to perform Sanger sequencing with mixed culture of an environmental sample? Or I need to isolate and purify single colonies for identification based on 16s gene sequencing employing the Sanger Chemistry?
If you try to sequence a mixture of various 16 S rDNA, you will end up with electropherograms showig a number of ambiguous base callings, without being able to assign them to a particular sequence or species. However, you don't need to cultivate each of the original organisms separately (this will usually be next to impossible). What you should do is amplify the 16 rDNA using primers hybridizing to conserved region of 16 S rDNA, generate a bank of by cloning the bulk amplified DNA and then sequence the clones arising from the bank, which will give you information about the strains present in the sample as well as their relative abundance.
I would suggest isolating and purify the bacteria.
eg.
if the mixed sample contains 50% bacteria A and bacteria B 50%, the PCR product might not from strain A or B, the sequence might contains some part from A and another part from B.
If the mixed sample contains 90% bacteria A, 5% bacteria B, and 5% bacteria C. the PCR fragment you got, almost from bacteria A.
Hi,
I would not recommend this approach. I would recommend a metagenomics approach with next generation sequencing.
Sincerely,
Jonathan H.
Thank you everyone for your answers. I knew about the metagenomics approach, but was not aware about the bulk amplified DNA method. In any case through all the three approaches suggested will pick the predominant forms at first place and that is the objective of the whole experiment. Isolation and purification for sure is cumbersome and time taking and would also enrich an organism with otherwise lower abundance.
The entire point of Sanger is the assumption that you have one type of template going into the reaction. Yes, I know that some folks use Sanger to look for SNPs or other small changes, but it's not designed for that purpose.
Growing single colonies will bias your experiment towards what samples grow well in culture.
Just do a next-gen library and sort out the species with bioinformatics. That way you eliminate the bias from culturing and you can estimate relative abundance.
Good luck!
DNA–DNA hybridization is the gold standard for identifying bacterial species . Because of the complexity of DNA–DNA hybridization, 16S rRNA gene sequencing is used as a tool to identify bacteria at the species level and assist with differentiating between closely related bacterial species
No, if you do pcr on mixed bacterial species you have to clone the pcr product into plasmids first and then do the sanger sequencing on each clones.
I would recommend do metagenomic analysis because it gives you the chance to determine a wide range of organisms.
- Badges
- Science topic