View source for Sequence Alignment for Phylogenetic Analysis

== Locate Sequences and Generate FASTA File ==
* The easiest way to find sequences is to start with a seed sequence then do BLAST searches restricting to RefSeq and the species of interest.
* To find a seed sequence start with NCBI Gene, then find the first Refseq mRNA (should start with NM) then click on that and find the protein (should start with NP)
* Paste that into your FASTA file (see next section) and name accordingly.
* Paste that sequence or its NP id into [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome NCBI Protein Blast].
* Set the parameters to:
** Database: Reference Proteins (refseq_protein)
** Organism: Start with mouse (''Mus musculus'') or human (''Homo sapiens''), depending on your goal consider adding zebrafish (''Danio rerio''), ''Drosophila melanogaster'', chicken (''Gallus gallus'') and ''Caenorhabditis elegans''

=== Generating a FASTA File===
* FASTA format is described [https://zhanglab.ccmb.med.umich.edu/FASTA/ here], and [https://en.wikipedia.org/wiki/FASTA_format here] you need each sequence to start with a >SEQUENCENAME followed by a return and then the sequence, in this case the protein sequence.  An example of a FASTA file would be:

<code>
>SEQUENCE_1

MTEITAAMVKELRESTGAGMMDCKNALSETNGDFDKAVQLLREKGLGKAAKKADRLAAEG

LVSVKVSDDFTIAAMRPSYLSYEDLDMTFVENEYKALVAELEKENEERRRLKDPNKPEHK

IPQFASRKQLSDAILKEAEEKIKEELKAQGKPEKIWDNIIPGKMNSFIADNSQLDSKLTL

MGQFYVMDDKKTVEQVIAEKEKEFGGKIKIVEFICFEVGEGLEKKTEDFAAEVAAQL

>SEQUENCE_2

SATVSEINSETDFVAKNDQFIALTKDTTAHIQSNSLQSVEELHSSTINGVKFEEYLKSQI

ATIGENLVVRRFATLKAGANGVVNGYIHTNGRVGVVIAAACDSAEVASKSRDLLRQICMH
</code>

* Save sequences in notepad, [https://notepad-plus-plus.org/ notepad++] or [https://www.sublimetext.com/ sublime] (not Word) as a <FILENAME>.fasta file.
* Sequence names cannot have spaces.  Generally its better to name it as '''mm_Gdf15-NM_004864.4''' where mm indicates mouse, Gdf15 is the gene name and NM indicates a [https://www.ncbi.nlm.nih.gov/refseq/ RefSeq mRNA].  If there are multiple mRNA's for the gene, name them

== Create Multiple Sequence Alignment using CLUSTAL Omega ==

* CLUSTAL Omega is available at https://www.ebi.ac.uk/Tools/msa/clustalo/
* Select output format NEXUS to import into Mr Bayes or PHYLIP format to import into PhyoBayes
* Generate phlogenetic trees with [http://megasun.bch.umontreal.ca/People/lartillot/www/download.html PhyloBayes] or  Mr. Bayes [[Using Mr Bayes to For Phlyogenetic Analysis]].  

=== PhyloBayes Analysis ===

* Mark in your notes the software version used.
* The PhyloBayes manual can be found [http://megasun.bch.umontreal.ca/People/lartillot/www/phylobayes4.1.pdf here].