I have constructed a phylogenetic tree using Mega6 software as enclosed find the attachment please. The tree represents different bacterial DNA sequence isolated and amplified using 16SRNA, however, I noticed that I am having Staphylococcus equorum everywhere in the tree? and I have been suggested to edit or correct any errors in the output sequence. Anyone have an idea how to perform that? and which software would potentially do that? Many thanks in advance
Regards
Mohammed
I have a Standard Operating Protocol that I wrote for my students using universal 16S rRNA sequence analysis (after PCR) to 'clean up' the sequence obtained from our core facility (using *.ab1 files) with MEGA5 software (MEGA ver. 6 is now available), basically...we examine sequence from PCR in both forward and reverse directions, clean up the ambiguous sequence on both ends of both reaction products by examining the sequencing 'chromatogram' of colored peaks (removing bad reads that would give ambiguous results), and then complement the remaining 'good sequence reads' and make a consensus. I always do that instead of taking the computer derived sequence from *.ab1 files. At least in my mind, it leaves me with cleaner sequences, perhaps because I may be more conservative in the sequence that we select to use. Maybe there is a better way to do this, but this is what I came up with :-). Enjoy.
Your tree figure has meaningless equal branch lengths (cladogram) rather than branch lengths proportional to distances, so we can only guess the type of problem that might have created the mis-classification or grouping of S. equorum sequences with sequences from many other bacteria. Building a phylogenetic tree wiil not produce an "output sequence", so I am not sure what you are asking.
I suspect that your multiple sequence alignment file fed into the tree building process in MEGA was not correctly aligned. What program did you use for alignment? CLUSTAL is usually acceptable for 16S of related bacteria, but you might also try MAFFT or other programs if CLUSTAL is failing.
http://mafft.cbrc.jp/alignment/server/
http://www.ch.embnet.org/software/ClustalW.html
Thanks to you all for your helpful comments, since this is first time to make a phylogenetic tree. @Artur Burzynski, I didn't do the sequencing we sent our samples to the sequencer and we got them as output data, then we blasted them using 4 peak tool, and NCBI wbesites to identify the gene sequence. Regarding to the (bootstrap), if you mean confidence interval (CI) as far as you know you can't show it in a circle phylogenetic tree, I have another image shows the traditional tree has the IC labeled on it.
@Brian Thomas Foley, Many thanks for your comment, I have used both Mega 6 and clustalW2 firstly the sequences were aligned and then sent to phylogenetic construction site. But I notice in ClustalW2 site that they only show the accession number of the gene rather the name of the strain.
The second tree you show, with meaningful branch lengths and a scale bar at 0.2 (indicating that the distances between your sequences are greater than 2.0, root to each tip greater than 1.0) meaning that each base is estimated to have mutated 2 times. Whereas I find for example that Stenotrophomonas maltophilia HQ224661 is 74% identical to Brevibacterium GU968479. So, it seems that your sequences were not correctly aligned to each other.
I have never observed CLUSTAl or other protams to rename my sequences when performing a multiple sequence alignment. If I feed in sequences named by species, I get back the same names. However, it is critical not to have charachers such as "(" or ":" in the sequence names, when building a tree, because those characters are used in the NEWICK tree format.
I am attaching here a multiple sequence alignment of a few 16S rRNA sequences plus a tree built from them. I aligned the sequences with CLUSTAL-W which cannot do a good job across this range of diversity of 16S sequences. There are regions of the alignment which should either be improved, or discarded prior to building a reliable tree. But the point is that distances across this tree are closer to 0.5 (still a large distance) than to 2.0 (which indicates the sequences are either misaligned or too distant from each other to be aligned reasonably).
Again many thanks to Peter M Muriana & Brian Thomas Foley who really makes it easy to follow their instruction and acheive the required task. Thanks to
I found a nice multiple sequence alignment of bacteria 16S rRNA sequences at the EMBL RFAM database:
http://rfam.xfam.org/family/RF00177#tabview=tab2
However, when I downloaded the alignment, the sequence names were not very informative to me. They included the accession number but not the Genus and species name.
>AY138309.1/1-1552
So, I used the sequence alignment renaming tool: http://veb.lumc.nl/SNAD/
to change the names to genus.species.accession and I am uploading that renamed alignment here. I am a little disappointed that the alignment has many uncultured environmental samples, and no solid information as far as I can tell on how the sequences were selected for inclusion in this alignment. It looks like it might be just one sequence per genus, of some sampling of bacterial diversity, but I don't see things like E. coli/Shigella/Salmonella in it.
There is a recent review in Nature of bacterial classification using 16S sequences here: http://www.nature.com/nrmicro/journal/v12/n9/full/nrmicro3330.html
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