View source for Potential Gene Across-Species Phylogenetic Analysis with Mr. Bayes

== Potential Gene Across-Species Phylogenetic Analysis with Mr. Bayes ==



=== Make databases of species genome FASTA files ===
<pre>
w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/makeblastdb -title testdb -dbtype nucl -in bb_latest_assembly.fasta
</pre>
<pre>
w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/makeblastdb -title testdb -dbtype nucl -in VVgenome
</pre>
<pre>
w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/makeblastdb -title testdb -dbtype nucl -in rice_communisTIGR_castorWGS_release_0.1.assembly.fsa
</pre>
<pre>
w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/makeblastdb -title testdb -dbtype nucl -in fragaria vescachr.fna
</pre>

=== Search for desired gene from Genbank ===

From species most closely related to target studied species (Vinis vinifera- Vaccinium corymbosum)<br>
[http://www.ncbi.nlm.nih.gov/gene/100256566 VINST1]

=== Download FASTA file of nucleotides ===

[http://www.ncbi.nlm.nih.gov/nuccore/NW_002240906?report=fasta&from=206721&to=208614 VINST1 nucelotides]

=== BLASTn FASTA file of gene against databases of species sequences ===
<pre>
w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/blastn -query VINST1.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"
</pre>
<pre>
w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/blastn -query VINST1.fasta -db VVgenome -outfmt "7 qacc sacc evalue qstart qend sstart send"
</pre>
<pre>
w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/blastn -query VINST1.fasta -db rice_communisTIGR_castorWGS_release_0.1.assembly.fsa -outfmt "7 qacc sacc evalue qstart qend sstart send"
</pre>
<pre>
w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/blastn -query VINST1.fasta -db fragaria vescachr.fna -outfmt "7 qacc sacc evalue qstart qend sstart send"
</pre>

=== Copy and paste top BLASTn hit for each species with FASTA headings into [http://www.ebi.ac.uk/Tools/msa/muscle/ MUSCLE window] ===
Run MUSCLE alignment

=== Copy alignments into simple NEXUS text file ===
 
Formatted below with suffix .nexus<br><br>
ntax is the number of taxa (sequences aligned)<br><br>
nchar is the number of characters of each alignment<br><br>

<pre>
#NEXUS

begin data;
	dimensions ntax=4 nchar=1200;
	format datatype=dna interleave=nogap=-;
	matrix <br>
	Vaccinium_corybosum AGTCGCGCTAGCGCTG…ATGCTCGGTAGATCG
	Vinis_vinifera	    AGTCGCGCTCGCGCTG…ATGCTCGATAGATCG
	Fragaria_vesca	    AGTCGCGCTCGCGCTG…ATGCCCGATTGATCG
	Ricinus_communis    AGTCGCGCTCGCGCTG…ATGCTCGATAGATCG
	;
end;

(shortened for demonstration, named VINST1.nexus)
</pre>

=== Download [http://mrbayes.csit.fsu.edu/download.php MrBayes] ===
Unpack .sit file<br>
Place .nexus file created above in same directory as MrBayes executable

=== Open MrBayes executable ===

Execute .nexus file in MrBayes executable window:
<pre>
execute VINST1.nexus
</pre>
Set evolutionary model:
<pre>
lset nst=6 rates=invgamma
</pre>
Set number of samples:
<pre>
Mcmc ngen=10000 sample freq=10
</pre>
Summarize the parameter values (burnin=25% of your samples):
<pre>
Sump burnin=250
</pre>
Summarize and visualize the trees:
<pre>
Sumt burnin=250
</pre>