Sequence Alignment for Phylogenetic Analysis: Difference between revisions

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 == 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===
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 * 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
+* 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 ==