Building maximum likelihood phylogenetic tree using BUSCO genes

In this guide, we will explain how to get the species phylogenetic tree using just the draft genomes (genomes that still don’t have the annotations yet). For data, we will use the sequenced legume genomes. You will need the following programs installed and access to a large cluster (we use Condo in this exercise).


Like mentioned before, we will use the sequenced legume genomes that are available on NCBI (both RefSeq and GenBank). Since it is most likely that the new genome you’re working won’t have good gene predictions, we will use the BUSCO genes to construct phylogenetic tree. BUSCO (Benchmarking Universal Single-Copy Orthologs) are ideal for this because, these genes are found in a genome in single-copy are evolutionarily sound. There are 1,440 BUSCO’s for embryophyta profile, which will be a good sample size for building phylogenetic tree in reasonable amount of time.

Dataset : At the time of writing this tutorial, there were 38 genomes in NCBI belonging to Legumes group (Fabaceae). Out of these 38, 17 genomes have chromosome level assemblies, 11 genomes as scaffolds, and 10 genomes as contigs. See table 1 for more information on these genomes.

Table 1: Legume genomes used in this study.

Organism SpeciesTaxid RefSeq_category Genbank ScaffoldN50
Arachis duranensis (eudicots) 130453 representative genome GCA_000817695.2 1,068,268
Arachis duranensis (eudicots) 130453 na GCA_001687015.1 149,039
Arachis ipaensis (eudicots) 130454 representative genome GCA_000816755.2 6,169,993
Cajanus cajan (pigeon pea) 3821 representative genome GCA_000340665.1 555,764
Cajanus cajan (pigeon pea) 3821 na GCA_000230855.2 5,341
Cicer arietinum (chickpea) 3827 representative genome GCA_000331145.1 697,963
Cicer arietinum (chickpea) 3827 na GCA_000347275.3 74,024
Cicer arietinum (chickpea) 3827 na GCA_002896005.1 232,601
Cicer echinospermum (eudicots) 90897 representative genome GCA_002896215.1 206,896
Cicer reticulatum (eudicots) 90898 representative genome GCA_002896235.1 109,263
Glycine max (soybean) 3847 representative genome GCA_000004515.3 48,577,505
Glycine max (soybean) 3847 na GCA_002905335.1 15,020,773
Glycine max (soybean) 3847 na GCA_001269945.2 42,937
Glycine soja (wild soybean) 3848 representative genome GCA_002907465.1 4,430,511
Glycine soja (wild soybean) 3848 na GCA_000722935.2 404,776
Lotus japonicus (eudicots) 34305 representative genome GCA_000181115.2 25,054
Lupinus angustifolius (narrow-leaved blue lupine) 3871 representative genome GCA_001865875.1 702,610
Lupinus angustifolius (narrow-leaved blue lupine) 3871 na GCA_002285895.2 30,409,451
Lupinus angustifolius (narrow-leaved blue lupine) 3871 na GCA_000338175.1 15,485
Medicago truncatula (barrel medic) 3880 representative genome GCA_000219495.2 49,172,423
Medicago truncatula (barrel medic) 3880 na GCA_002024945.1 12,848,239
Medicago truncatula (barrel medic) 3880 na GCA_002251925.1 575,111
Medicago truncatula (barrel medic) 3880 na GCA_002251935.1 859,387
Medicago truncatula (barrel medic) 3880 na GCA_002251955.1 503,143
Phaseolus vulgaris (eudicots) 3885 representative genome GCA_000499845.1 50,367,376
Phaseolus vulgaris (eudicots) 3885 na GCA_001517995.1 433,759
Trifolium pratense (eudicots) 57577 representative genome GCA_900079335.1 22,682,783
Trifolium pratense (eudicots) 57577 na GCA_000583005.2 2,432
Trifolium subterraneum (eudicots) 3900 representative genome GCA_001742945.1 287,605
Trifolium subterraneum (eudicots) 3900 na GCA_002003065.1 1,510
Vicia faba (fava bean) 3906 representative genome GCA_001375635.1 1,723
Vigna angularis (adzuki bean) 3914 representative genome GCA_001190045.1 1,292,063
Vigna angularis var. angularis (adzuki bean) 3914 na GCA_001723775.1 8,174,047
Vigna angularis var. angularis (adzuki bean) 3914 na GCA_000465365.1 21,697
Vigna radiata var. radiata (mung bean) 157791 representative genome GCA_000741045.2 25,360,630
Vigna radiata var. radiata (mung bean) 157791 na GCA_001584445.1 683,756
Vigna radiata (mung bean) 157791 na GCA_000180895.1 228
Vigna unguiculata subsp. unguiculata (cowpea) 3917 representative genome GCA_001687525.1 7,412

The data can be downloaded from using the commands:

Download the Data

For downloading, you can do it directly from the NCBI website. To do so, go to NCBI genomes page for Fabaceae, click on Download Assemblies, select the GenBank as source database and Genomic FASTA as the file type, followed by Download

To easily indetify these files, we will rename them.

Now we are all set to begin the analysis!

Run BUSCO to predict conserved genes

Running BUSCO is straight forward. The BUSCO for plants have 1,440 total genes and running BUSCO will predict those genes from the genomes. Of course, the number will vary depending on how complete the genomes are but we can use the common genes that were found in 3 or more genomes to build the gene tree and then use it for inferring the species tree.

For running BUSCO we will use This script will make it easier to run on several genomes by limiting the complexity and standardizing analyses to best parameters. The script can be found here

To run this in parallel we will use a for loop to generate commands for each species.

for file in *.fasta; do
echo "./ $file";
done >> busco.cmds

Next, generate multiple PBS scripts to run them on a cluster. For Condo the script can be used (script can be obtained from our repo here.

After this step, you should have 21 BUSCO submission scripts. Submit them to the cluster using for loop:

for file in busco_?.sub; do
sbatch $file;

Optional step: Get the BUSCO plot for your genomes

Copy the short summary files from BUSCo run directories to another directory for getting the BUSCO plots

mkdir busco_summaries
for file in $(find . -type f -name "short_summary_*"); do
cp $file ./busco_summaries/;

Generate the BUSCO plot using the script in the BUSCO package

python3 ${BUSCO_HOME}/ -wd busco_summaries

This should generate busco_figure.R and busco_figure.png files.

Extract complete BUSCO genes

Next, we need to find all the “complete” BUSCO genes predicted from all of the genomes. Since 1,440 genes have unique ids, we can quickly get a list of all gene names that are complete (from BUSCO logs) and use them for downstream analysis.

for file in $(find . -name "full_table_*.tsv"); do
grep -v "^#" ${file} | awk '$2=="Complete" {print $1}' >> complete_busco_ids.txt;

To filter out the complete BUSCOs that are only present less than 3 genomes, we can use uniq and awk commands

sort complete_busco_ids.txt |uniq -c > complete_busco_ids_with_counts.txt
awk '$NF > 2 {print $1}' complete_busco_ids_with_counts.txt > final_busco_ids.txt

Now let’s copy the genes (both nucleotides and amino acids) to different directory. Note that the gene names are just indetified as BUSCO id with either faa or fna extenstions. If you try to cooy them directly to a single directory, you will likely overwrite all the files and end up with only the last set of files. You can give them a unique name and then merge them all together, writing them to a single busco id file. before you do this, you will also need to edit the sequence name (fasta header) so that it includes organism identifier in it.

mkdir -p busco_aa
mkdir -p busco_nt
for dir in $(find . -type d -name "single_copy_busco_sequences"); do
  sppname=$(basename $(dirname $dir)|cut -f 2-3 -d "_" | sed 's/_/ /g');
  abbrv=$(echo $sppname | sed 's/\(\w\w\w\)\w*\( \|$\)/\1/g')
  for file in ${dir}/*.faa; do
    cp $file busco_aa/${abbrv}_${file}
    sed -i 's/^>/>'${abbrv}'|/g' busco_aa/${abbrv}_${file}
	cut -f 1 -d ":" busco_aa/${abbrv}_${file} | tr '[:lower:]' '[:upper:]' > busco_aa/${abbrv}_${file}.1
	mv busco_aa/${abbrv}_${file}.1 busco_aa/${abbrv}_${file}
  for file in ${dir}/*.fna; do
    cp $file busco_nt/${abbrv}_${file}
    sed -i 's/^>/>'${abbrv}'|/g' busco_nt/${abbrv}_${file}
	cut -f 1 -d ":" busco_nt/${abbrv}_${file} | tr '[:lower:]' '[:upper:]' > busco_nt/${abbrv}_${file}.1
	mv busco_nt/${abbrv}_${file}.1 busco_nt/${abbrv}_${file}  done

This will convert the spp name to an abbreviation (from Vicia_faba to Vicfab) and edit the fasta header to include it in the name (eg. >EOG09360J1D to >VICFAB|EOG09360J1D). Next step is to generate single file for each of the BUSCO id. We will do this with a simple bash loop. Remember the file final_busco_ids.txt we created previously? we need that for this purpose:

while read line; do
cat ??????_${line}.faa >> ${line}_aa.fasta;
cat ??????_${line}.fna >> ${line}_nt.fasta;

As we won’t be needing the individual fasta files (because we now how a single file for each BUSCO gene that contains complete sequence from all the species), let’s go ahead and delete them (alternatively, you can move them to another directory as backup copy, just in case)

rm ??????_EOG09360???.f?a


NOTE: we will only use the peptide sequence for building tree here, but you can easily change this to do the same with nucleotide sequence as well. We will use the found here and found here for running alignments.

for file in *_aa.fasta; do
echo "./ $file";
done > guidence.cmds 200 guidence.cmds
for sub in guidence_*.sub; do
sbatch $sub;

Once the alignment completes, you will see a folder (same name as the input sequence but without fasta extension) and in each directory will have many files. we will only be needing Seqs.Orig.fas.FIXED.Without_low_SP_Seq.With_Names file for the next step. You also need to make sure that the alignment completed successfully by checking if there is any error message in the log file.

Next, let’s copy the final alignment file to a different directory where we will run the RAxML.

mkdir raxml_aa
# from the directory where you ran guidence from
for aln in $(find . -name "Seqs.Orig.fas.FIXED.Without_low_SP_Seq.With_Names"); do
busco=$(dirname $(basename $aln));
cp $aln ./raxml_aa/${busco}.aln;

Gene tree reconstruction

Gene phylogenetic tree will be reconstructed with maximum likelihood (ML) approach using RAxML program. We will use a rapid bootstrap analysis and search for the best-scoring ML tree with 1000 bootstrap replicates. Best protein model with respect to the likelihood on a fixed, reasonable tree will be automatically determined using the PROTGAMMAAUTO option of RAxML (other option is to test which evolutionary model fits your data by running prottest or other similar programs). Arabidopsis thaliana will be the outgroup.

Again, we will use the (found here) and scripts for running the jobs:

cd raxml_aa
for aln in *.aln; do
echo "./ $aln AA"
done > raxml.cmds 200 raxml.cmds
for sub in raxml_*.sub; do
sbatch $sub;

Once complete, you will have RAxML_bestTree.EOG09360??? file that contains the best scoring tree (not the bootstrap consensus tree). The branch scores represent the likelihood score. If you want to get the bootstrap support trees you should be using RAxML_bootstrap.EOG09360??? file that will have 1000 bootstrap replicates to generate a consensus tree. Here, we will use the ML tree.

To generate a single file with ML trees for all BUSCO genes:

cat RAxML_bestTree.EOG09360??? >> busco_genes_ML.tree

Species tree reconstruction

Before we proceed with the species tree, we need to fix the title (or the taxa name) in the gene trees. As even single tax in these gene trees are identified as genspp|buscoid, they all have unique ids and it will be considered as seperate taxa. We don’t want this as we only have 22 tax here. For this, we need to remove the buscoid from the name (i.e, convert names that look like this LOTJAP_03I2 to just LOTJAP). We will use the sed command for that purpose.

# create a file with all the abbreviation's
# pick any one RAXML best tree from teh raxml_aa folder
grep -oE "......_...." RAxML_bestTree.EOG093603IM |cut -f 1 -d "_" > capabrv
# edit the merged ML tree file to replace it
while read line; do
sed -i 's/'${line}'_....:/'${line}':/g' busco_genes_ML.tree;

As a last step, we will generate a species tree using the ASTRAL coalescent-based species tree estimation program. ASTRAL is the fast method for estimating species trees from multiple genes which is statistically consistent, and can run on multiple gene trees efficiently.

java -jar ${ASTRAL_HOME}/astral.jar --input busco_genes_ML.tree --output busco_genes_ASTRAL_spp.tree

You will get a newick format tree file that can be uploaded to EvolView to generate publication quality graphics (tree). It can be accessed from this link.