It depends on the depth of how much you want to know about the fine details, 16S is a good choice if your goal is to have the overall diversity, but if you want to distinguish between close related strains of the same species it won't be enough, that's the goal of MLST approaches. In the deep issues about this try to read this one: http://www.sciencemag.org/content/323/5915/741.long

16 S rRNA gene Sequencing

I would recommend to do both methods.

Dear Ajar,

16S not good for species identification, it works upto genous level

Depending on the genus, for example, to identity species of Bacillus it is not useful to use the 16rRNA, because it's not enough to differenciate. Then, it's better to use housekeeping genes. But in my opinion, it depends which microorganism you want to identify. It's interesting try first the 16S and after, it's necessary, use other primers.

whats about Pseudomonas and Enterobacter genus?

It depends on the depth of how much you want to know about the fine details, 16S is a good choice if your goal is to have the overall diversity, but if you want to distinguish between close related strains of the same species it won't be enough, that's the goal of MLST approaches. In the deep issues about this try to read this one: http://www.sciencemag.org/content/323/5915/741.long

yup, nice comment Luis and Armando but lack of variability of the 16S rRNA gene sequence, more then one copy number and copy number varies per species not favor 16S rRNA for genus also and on the other hand MLSA have single copy number and gare protein encoding genes, the data generated from these markers is more readily interpreted in an evolutionary framework. but tell me the cut value of MLSA for species or novel species identification like if the 16S rRNA sequence of any strain show less then 97% homology consider to be novels what about Housekeeping gene???????

97% identity (not homology) is the cut-off for the species, which seems artificial for some of us... This value come from correlating the DNA-DNA association cut-off of 70% (previous of sequencing) when comparing different species of bacteria, which is a working definition for species, but not so sure about its real biological relevance. With MLST what you define are alleles and Sequence Types there is not such thing like a cut-off value.. check out recent MLST publications and the MLST DBs to get into it. Maybe it would be better to try with phylogenetic distances to describe novel groups when using coding sequences.

agreed Luis, if DNA-DNA hybridization is good enough for species identification because i read few paper where clearly mention that DNA–DNA hybridization is a technically thought-provoking, laborious and time-consuming method, it is not applicable to the study of non-culturable bacterial strains in the biosphere.Moreover, the technique has the drawback that hybridization values of 50% or less are less informative and therefore DNA–DNA hybridizations are not suitable for the estimation of genetic distances between distantly related species.

new query is that which is the best for species identification or species novelty housekeeping genes (MLSA) or DNA-DNA hybridization ???

MLSA, for sure ;)

Yup, we can compare the sequences of housekeeping genes for bacterial identification and classification or depend on phylogenetic analysis ??

16S rRNA is proven method.

16SrRNA

Dear Pattnaik,

if you could explain your answer in which regard 16S rRNA proven, novel species identification, species description or phylogeny analysis ?????

I think it depends on the expected type of bacteria you're looking for. In some cases specially in case of Pseudomonads, it has been proven that 16SrRNA are not good accurate to identify the species. Thus, MLSA can give more reliable result rather than 16SrRNA.

Dear mahdi, i agreed with your opinion, pseudomonas well characterized by MLSA at species level

It really just depends on your application. 16S and DNA-DNA Hybridization are both standard practice, which is I am sure what the above poster meant when he said it is a proven method. Here's a link to an article describing how to isolate and identify bacterial DNA from human samples using their 16S RNA.

http://goo.gl/XoT5K

Here's a link to a study comparing MLSA to 16S and DNA-DNA hybridization. They found MLSA to be more robust, but again the best method really depends on your application.

http://ijs.sgmjournals.org/content/58/1/200.full

Dear Fogarty,

Thanks, i have read this publication and i got your point, i want to do comparitive study of MLSA and 16S from rhizoshphere source.

You can do 16S rRNA analysis but remember some microorganisms might have two of these which might be confusing. Also some strains of Bacillus have exactly the same 16S rRNA sequence. So, once you do 16S rRNA analysis, do conduct analysis of Housekeeping gene sequences to be sure.

16S rRNA analysis should be sufficient for a general bacterial profiling, However, to identify bacteria species in deeper level, other analysis should be combined.

16S rRNA is fine. We obtained good results.

Dear Pezzolla,

if you got good results of 16S rRNA for species identification then its amazed me because 16S rRNA have multiple copy number in a strain so difficult to differentiate at species level but MLSA can do this because these genes have single copy number.

16S rRNA is fine.

I use the fAFLP methods.......also for root taxonomic study.

16S rRNA is really best choice....

Hi,

I have a related point so the followers of this question could help.

I have animal tissue samples with varying levels of DNA concentrations. I want to detect disease causing bacteria in them. I plan to run PCR using 16S rRNA primers on these samples to confirm the presence of general bacterial DNA that is sufficient for having a good PCR. Then I will run these 16S rRNA "positive" samples in a second PCR with the disease specific primers. I am trying to rule out that the negative PCR was not caused by not having sufficient bacterial DNA in the sample but it occurred because the disease causing bacteria was not present. Please let me know what do you think.