Abundance of phylogenetically coherent guilds can be determined in complex environments (complex communities), using a qPCR approach with specific primers targeting either the 16S rDNA gene or a chosen functional gene.
When applying these 2 methods to quantify ammonium oxidizing bacteria (or archaea), we observe - however - more than an order of magnitude difference in abundance (which we think cannot be explained by gene copy nr differences).
Has anyone else observed, documented or seen this documented? I am eager to hear from you.
Hey Barth, I suggest you clone your qPCR products to see wether the diversity was equally recovered. I am curious about the specificity and coverage of qPCR primers you used. Probably one of the primer sets was not able to amplify the most of the community so that the abundance was much lower. Nonetheless, the copy number of 16S rRNA gene is bigger than that of functional genes in most cases, which will also result in the divergence of quantification. Hope my words are helpful. Good luck
Coverage of the different genes in the environment would be one answer, but PCR bias is equally probable. Here (http://www.ncbi.nlm.nih.gov/pubmed/23757271) you will find a recent paper where we describe one such bias, which we termed "next base effect", where the first base downstream from the 3' end of your primer affects the over all amplification efficiency and also skew the relative abundance of sequences in the resulting libraries.
The fact described by you is very similar to finding around plants pathogen-related strains in proportion 10:1 (non-virulent : virulent). Plant pathogenic bacterium (Agrobacterium tumefaciens would be the best example) provides food to large population of relatives, that have nearly identical 16S and other taxonomic genes, but have no functional genes for parasitism.
Dear Fang Liu
In response to your suggestion, indeed - that is what we did - we cloned both the results from a 16S rRNA targeted qPCR and from an amoA targeted qPCR. The amplicons were NOT congruent in terms of the phylogeny they picked up - they were both literature derived, and according to our in silico testing good and similar in coverage. But experimental assessment showed differently - they were biased towards different groups. Just wondering if someone has published similar observations -- Barth
Hey Barth, did you mean your primers didn't amplify the targeting groups albeit the primers were well designed in silica? I am not an expertise in qPCR so I don't know anyone ever published similar observations. Maybe in this case, you need to increase the annealing temp and targeting length to ensure the specificity of primers. Are you using SYBR-GREEN kit? The specificity of primers does vary when dealing with complex templates. Maybe you can try Taqman kit which is based on probes. Good luck.
I would suggest you to consider a single copy gene. 16S rRNA gene is a multicopy gene and for phylogenetics rpoB gene which is a single copy gene is also very much used. See how the results behave. http://aem.asm.org/content/66/8/3376
- Badges
- Science topic
- Similar topics
- Agricultural Science
- Agronomy
- Crop Science