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#Constructing #Building  aPhylogenetic Tree using  16S rDNA sequence  There are two points during Phylogenetic Tree  What _is_ this thing? At this point, you have an organism in pure culture, but you don't know what it is. If you found an creature crawling on  the workflow where making a 16S phylogenetic tree may be useful. The first is after identification of candidate organisms by Sanger sequencing ground and  wanted to identify (or classify) it, you might look at it's morphology  and ask what it most _looks_ like. If it has six legs, you might guess it's some kind of  insect. If it has hard outer wings folded over its back, you might guess that it's some kind of beetle. If it also had antler-style horns on its head, you might  guess that it's some kind of stag beetle. If you don't have enough information available to guess what kind of stag beetle you have, then you have reached  the second is after assembly limit  of *taxonomic resolution* for your creature. With an unknown microbial species,  the genome. The process best way to identify it  is identical in both cases, but the additional length and improved quality to sequence one  of its genes (most people  use  thepost-assembly full-length  16S rRNA gene sequence may generate a better tree. The tree can be used for identification of gene) and ask what _it_ most looks like. With animal classification, the key features to examine are things like legs and wings and horns; with microbial  classification,  the candidate (e.g. is key features to examine are  the candidate found nucleotides in different positions  in a single species clade?), for naming DNA sequence. Fortunately, we have computer programs to help us make  sense  of the candidate (does it fall in DNA sequence information. Our preferred approach to identifying  a clade containing only members microbial species is to place the unknown sequence in the context  of that species, and other members a  phylogenetic tree  of known sequences. Building a phylogenetic tree from a 16S rRNA sequence is fairly straightforward, but  the species are not found outside that clade?), and for placement interpretation  of the organism into tree can be  a phylogenetic context. bit of an art. Here, we attempt to guide you through both. However, some complicated cases will require consultation with an expert in the field of  phylogenetics or systematics.  The outline of the workflow is to use the Ribosomal Database Project (RDP) to generate an alignment of the sequence with close relatives and an outgroup, following by cleanup of the RDP headers, tree-building with FastTree and viewing/analysis viewing/interpretation  of the treein Dendroscope.  Obtain the Full-Length 16S Sequence from the Assembly  (Skip this step if you are building the tree  using the 16S sequence from Sanger sequencing)  1. Go to RAST and sign in  On the “Jobs Overview" page, click on view details (under annotation progress) for the microbe you are working with.  2. Click on Browse annotated genome in SEED viewer (At the top of the page)  3. Click on Browse through the features of [organism name]  4. Under Function search for “ssurna” or “SSU rRNA”  (if it doesn't work at first then refresh the page)  5. Find the ssuRNA that is 1400-1800 bp in length (often Illumina assemblies also have fragments of 16S sequence that are only a few hundred bp long)  6. Click on the Feature ID for that sequence  7. Click on the Sequences tab (around the middle of the page )  8. Click on Show Fasta  9. Click on Download Sequences and save as a fasta file. Rename the file to something useful.  11. Double check the identity of the sequence at BLAST:  http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome  Obtaining Dendroscope.  ##Obtain  an RDP alignment The goal of this section is to obtain a 16S alignment from RDP that can be used to build a tree. The "art" is determining which sequences to put in the alignment. 1.  Go to rdp.cme.msu.edu 2.  Create an account 3.  Click on my RDP/login 4.  Upload the fasta file containing your 16s sequence 5.  Assign it a group name (this is what the program will label your sequence/organism). Choose this carefully since that will be the name on the final tree. Upload the fasta file (may take some time to process, status=P (pending) wait until A (aligned)  If it is taking awhile refresh the screen 6.  Click the "+" next to the sequence, to add it to your cart 7. Click on CLASSIFIER at the top of the page  8. Click on "Do Classification With Selected Sequences" button. This will show you a hierarchical view of the classification of your sequence (from Phylum to Genus.) You will use this information to find other sequences that you want to include in your alignment that you will use to build your phylogenetic tree.   8. Under "Data Set Options," Specify isolates as the Source, leave the rest as defaults    click on BROWSERS at the top of the page  Specify isolates as the Source, leave the rest as defaults  Click Browse   Browse Sequences