Hi,

I have to disagree with Stephen that 'any sequence not in the tree' would be a good outgroup. In general practice of phylogenetic reconstruction, one would want to use the most closely related sequence(s) outside the group of interest. Of course, there are other criteria that should be considered, e.g., the quality of the sequence(s) to be used as outgroups, the length of the sequences, i.e., are they long enough to use in alignments and comparisons with your sequences, etc? But, whenever possible, it is best to use the most closely related sequence(s) possible. In the case of the bacterial genera you mention, Bacillus spp. (Firmicutes) and Devosia spp., etc., (Proteobacteria), you are suggesting to present the phylogenetic relationships of a relatively wide range of bacterial taxa. But, I would argue that these are not so extensive that you should use archael sequences. If you propose to present all sequence data for the genera you mention in only one figure, Planctomycetes probably would be a good outgroup.

In any case, the question always is - what do you want to show? Usually, phylogenetic reconstruction is used to present the relative phylogenetic (and taxonomic) relationships of the bacteria in your study, with respect to their close relatives or with respect to important reference species or strains. The larger the phylogenetic tree is and the more extensive the bacterial taxa is in a phylogenetic analyses, the lower is the phylogenetic resolution between individual members. To try to solve that, I would argue that the best presentation of the diversity of bacteria that you are studying would be to present separate phylogenetic trees of the different genera or families for which you have sequence data. For example, if you want to present Bacillus spp., I would suggest to present your sequences within a spectrum of other Bacillus spp. Of course, Bacillus includes almost 300 validly published species and can be seen to encompass many different phylogenetic 'lineages'. You may want to select the relevant species of the lineages where your sequences cluster. Depending on the number and phylogentic diversity of the sequences you are analysing, you may want to present more than one tree. With the use of 'Supplemental Material' these days by journals, there is no excuse for not including as many figures and tables as you want to. In the case of Devosia spp., Serratia spp., Stenotrophomonas spp., Pseudomonas spp., etc., you have representatives of the Alphaproteobacteria Class and the Gammaproteobacteria Class. You may decide to present a figure with your sequence data and reference sequences from Proteobacteria, overall, or break it down into the different Classes. Again, I would recommend to present your data within the context of the respective genera and, possibly, representative species of closely related genera. Particularly, in the case of Serratia, you may want to include, as well, species of other genera within the Enterobacteriaceae. Pseudomonas is somewhat similar to Bacillus, in that it includes more than 200 species. Thus, you may need to select the species of the relevant phylogenetic lineages as the best way to show the relationships of the bacteria that you are analysing.

Whenever possible, you will want to include important 'reference' species in your phylogenetic analyses.

I hope these recommendations help. Best of luck with your research

@Edward : Thank you for appropriate explanation which is  elucidating why my few sequences considered as outgroup interfered with other clusters. My objective is grouping my  different set of sequences for confirming the identity and studying the relationships among them. However, I have chosen Microbacterium  sps as outgroup  (since in my group only one 16sRNA  sequence is present which matched 90% with Genbank ) Other than this, . I found two different Microbacterium  species and made a tree which gives good picture of grouping. Do I need to go further with Planctomycetes  sps now.( Any way I check with that too)

I learn that grouping will be more accurate  if the quality of sequences , lineages are appropriate .

@Stephen  I noticed that you have mentioned  bacterial phylum in your previous message. Thank you for that. 

Hi,

Apologies on the confusion of my wording. I would still argue that any phylum not already in a phylogenetic tree is not necessarily equal as an outgroup for clustering.

One way to identify potential outgroup sequences is by doing BLAST (NCBI) or FASTA (EBI) searches. As you go down the list of matches to your sequence data, you will eventually see bacterial species that are outside the genus or family or class, etc., of the taxon/taxa that you are interested in. Since the search matches are listed in order of decreasing similarity, you should be able to identify the bacterial species that could serve as reliable outgroup(s).

Thank you for the dialogue and best regards.

@Amit K G. I have used one of isolates from our group ( strain ) as a outgroup since it produced good branches for phylogeny analysis.

@N. Subash, the purpose of getting one tree for so diverse sequences (Bacillus sps, Devosia sps, Serratia sps,  Stenotrophomonas sp,  Pseudomonas sp) is not clear. As Edward Moore has noticed, each group has many species, and it is more reasonable to analyze isolates of each group among the closest relatives, including representatives of all known species within each genus, which contains your isolates of particular group.   

hello to all respectives, I was gone through all the answers. Now I ahve one question that if I am having sequence of both gram postive and nagetive bacteria. Than what would be the appropriate outgroup. As I am havin 26 sequence of 16srRNA region.

Neha Shukla maybe you can use aquifex as out-group for 16S