with 16S there are some roles to identify your isolates:

1- as much nucleotides you get from sequencing the better the identification it is.

you can get this by sequencing both limits, or by fragments.

2- with your 600 nucleotide it is sufficient to identify the genus level and sometimes the species level, the case of non "crowded" genus.

3- in case of some "crowded" genus such as Bacillus or Streptomyces it is really difficult to get beyond the genus level ( you just approach the species or sometimes the so called complex or group ) and in this case you say the most related species is ........ or belong to the group of ......

4- for the strain level, I believe, you should apply either sequencing of other genes or other biochemistry- immunology tests

* hope this helped, good luck.

with 16S there are some roles to identify your isolates:

1- as much nucleotides you get from sequencing the better the identification it is.

you can get this by sequencing both limits, or by fragments.

2- with your 600 nucleotide it is sufficient to identify the genus level and sometimes the species level, the case of non "crowded" genus.

3- in case of some "crowded" genus such as Bacillus or Streptomyces it is really difficult to get beyond the genus level ( you just approach the species or sometimes the so called complex or group ) and in this case you say the most related species is ........ or belong to the group of ......

4- for the strain level, I believe, you should apply either sequencing of other genes or other biochemistry- immunology tests

* hope this helped, good luck.

It is also important which part of 16S gene is your 600 bp fragment.

HI..

For complete identification you should need long stretch of sequence (16S RNA size is 1500 bp).

The sequencing should be done from both side (5' and 3' end) of template strand.

After the sequencing both sequence should paired, their ends should merged in between. other wise PCR artifacts creates problems, there will be a chimera problem.

Thanks you all

Depends on how many and which species you think you have in your sample and also which part of 16S you manage to amplify. Sometimes, very short fragments are still informative enough to distinguish between many species. There tools to test this. If you consistently amplify the same 'short' fragment of 16S, you could test whether it is informative or not using the sliding window analysis in the R package SPIDER (see here:LINK).

http://www.plosone.org/article/metrics/info%3Adoi%2F10.1371%2Fjournal.pone.0038215#usage

If I were you, I'd take a look at

http://cge.cbs.dtu.dk/services/MLST/

I did quite some of 16S rDNA for identification and 1500 bp is the usual length, as is mentioned mostly as partial sequence, and you would find this length in most of the publications. Normally one would go for two end sequencing and then assemble the whole length. For your 600 bp limit, then for better identification I would design a third primer in between that spans the length of DNA so that I could make a full contig.

Dear Dr. Arif:

I also did the 16SRNA sequencing and got more than 1450 bp sequence.

then I blast it in NCBI against 16S RNA Bacteria and Archea deatbase.

In addition I also analysed top 30 sequence in MEGA 4.

My query is; on what basis phylogenetic trees are analysed or what are the criteria to analyse phylogenetic tree,

If you like I can send you phylogenetic tree along with result.

I would be very grateful to you. Thank you very much

Dear Anand

its difficult for me to explain in detail how phylogentic trees are generated, but in short, trees are based on calculating the distances in relation to the number of mutations from each other. so lesser the mutations, the closer they are. and while you use mega, you can see there are different methods to use. There is a nice article in 2008 about evolutioanry trees, I think it would be helpful

http://www.cbs.dtu.dk/courses/27615.mol/pdf/understanding_evo_trees.pdf

Here is a recent course at coursera, this wil be really helpful for your particular question.

https://www.coursera.org/course/bioinfomethods1

GL

Is it necessary that I use both forward and reverse primer for sequencing 16s RNA gene in bacteria?

For the PCR step you should use both primers, then do sequencing PCR

refer to the link for the sequencing PCR

https://www.researchgate.net/post/How_do_I_directly_perform_sequencing_PCR_on_a_PCR_product_without_going_through_cloning

I plan to use 27F primer, what length will the sequence be?

you should use the R primer such as 1492R with 27F to get the ~1500 pb 16s rDNA

27F and 1492R are universal primers. You can use this primer set to get the sequence information of about 1500 bp. You can request the company to join the pair to give complete sequence.

For species identification, better the length OF SEQUENCE , BETTER IS THE IDENTIFICATION

Yes, it is publishable,but which gene it is and how much informative is it?

How can I make sure that theses primers : 1492R with 27F are suitable for my organism? any suggestion on ways to check?