How to isolate genes from whole genomes for phylogenetic tree analysis?

Question

I have 446 whole Klebsiella Pneumoniae genomes I want to build a phylogenetic tree from. After reading about constructing phylogenetic trees it seems the only option for large numbers of genomes is to isolate a gene with low variability from generation to generation and use this gene to build a tree. For example Lars Jensen recommends using "16S rRNA [or] all ribosomal-protein-coding genes" https://www.biostars.org/p/1930/. What program isolates these genes of interest from the whole genome fasta files and can put them into a multiple alignment file? Or outputs them in a formate ready for a multiple alignment program such as Muave? The reason I say a multiple alignment file is because this it the type of file most phylogenetic tree programs take (I.E. clonalframe).

Answer 1

There are a lot of ways to do this. I suggest using Prokka/Roary to produce a core genome alignment. There's a useful tutorial on the Roary website:

for file in *
do
    prokka --kingdom Bacteria --outdir "${file%%.*}"  --genus Listeria --locustag "${file%%.*}" "$file"
    mv "${file%%.*}"/PROKKA_07052017.gff GFF/"${file%%.*}".gff # use current date
done
roary -f Alignment -e -n -v GFF/*.gff

Alignment/core_gene_alignment.aln can be used as input for phylogenetic analyses

Answer 2

Extract desired gene sequences using standalone blast

Simply provide a reference database with your desired output. Set up your command and away you go. You can set the search up with a for loop for a batch of sequences. Command may look like

for f in *.fasta; do
   f=$(basename $f .fasta)
   blastn \
   -outfmt "6 sseqid qseq %" \
   -query $f.fasta \
   -subject reference.fna \
   > out/$f.fas
done

Watch the output as blast will spit out the gene as detected in + or - sense. If you want to only gather positive sense use -strandoption. The default output I have here is tab output which requires a few sed commands to make into fasta.

sed -i \
-e 's/\s*$//g' \
-e 's/^/>/g'  \
-e 's/\s\+/\n/g' \
*.fas 

Online alignment servers are an easy way to align small datasets e.g. EBI