Potential Gene Across-Species Phylogenetic Analysis with Mr. Bayes

Make databases of species genome FASTA files

w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/makeblastdb -title testdb -dbtype nucl -in bb_latest_assembly.fasta

w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/makeblastdb -title testdb -dbtype nucl -in VVgenome

w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/makeblastdb -title testdb -dbtype nucl -in rice_communisTIGR_castorWGS_release_0.1.assembly.fsa

w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/makeblastdb -title testdb -dbtype nucl -in fragaria vescachr.fna

Search for desired gene from Genbank

From species most closely related to target studied species (Vinis vinifera- Vaccinium corymbosum)
VINST1

Download FASTA file of nucleotides

VINST1 nucelotides

BLASTn FASTA file of gene against databases of species sequences

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"

w10120:Desktop jamimms$ /usr/local/ncbi/blast/bin/blastn -query VINST1.fasta -db VVgenome -outfmt "7 qacc sacc evalue qstart qend sstart send"

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"

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"

Copy and paste top BLASTn hit for each species with FASTA headings into MUSCLE window

Run MUSCLE alignment

Copy alignments into simple NEXUS text file

Formatted below with suffix .nexus

ntax is the number of taxa (sequences aligned)

nchar is the number of characters of each alignment

#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)

Download MrBayes

Unpack .sit file
Place .nexus file created above in same directory as MrBayes executable

Open MrBayes executable

Execute .nexus file in MrBayes executable window:

execute VINST1.nexus

Set evolutionary model:

lset nst=6 rates=invgamma

Set number of samples:

Mcmc ngen=10000 sample freq=10

Summarize the parameter values (burnin=25% of your samples):

Sump burnin=250

Summarize and visualize the trees:

Sumt burnin=250

Potential Gene Across-Species Phylogenetic Analysis with Mr. Bayes

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