Finding orthologs using OrthoMCL program

This is the first section of Phylogenomics chapter, where we predict orthologs from the genes of whole genomes. Although, there are several methods to predict orthologs, we will primarily use OrthoMCL program. Since orthoMCL depends on MySQL, we will use Singularity containers for running the MySQL server (and OrthoMCL). We will use many different species of Plasmodium as an example dataset for this exercise.

Following is the outline for this chapter:

  1. Obtaining the data
  2. Cleaning and formating sequences
  3. Running orthoMCL program
  4. Formatting and processing results (for downstream analysis)

1. Downloading data

At the time of writing this tutorial, there were 13 RefSeq assemblies available for Plasmodium spp. on NCBI (with chromosomal level assembly). We will use them all for finding orthologs. We will only need the protein sequences for this section. This step may vary if you want to use another dataset or custom dataset so you may have to change the below commands accordingly.

Downloading Proteins

Download it and extract the archive using tar command, and explore the contents.

tar xf genome_assemblies.tar
cd
grep -c ">" *.faa

Following is the summary:

Filename Species Sequences
GCF_000002415.2_ASM241v2_protein.faa Plasmodium vivax Sal-1 5,392
GCF_000002765.4_ASM276v2_protein.faa Plasmodium falciparum 3D7 5,392
GCF_000006355.1_ASM635v1_protein.faa Plasmodium knowlesi strain H 5,101
GCF_000321355.1_PcynB_1.0_protein.faa Plasmodium cynomolgi strain B 5,716
GCF_000524495.1_Plas_inui_San_Antonio_1_V1_protein.faa Plasmodium inui San Antonio 1 5,832
GCF_000709005.1_Plas_vinc_vinckei_V1_protein.faa Plasmodium vinckei vinckei 4,954
GCF_000956335.1_Plas_frag_nilgiri_V1_protein.faa Plasmodium fragile 5,672
GCF_001601855.1_ASM160185v1_protein.faa Plasmodium reichenowi 5,279
GCF_001602025.1_ASM160202v1_protein.faa Plasmodium gaboni 5,354
GCF_001680005.1_ASM168000v1_protein.faa Plasmodium coatneyi 5,516
GCF_900002335.2_PCHAS01_protein.faa Plasmodium chabaudi chabaudi 5,158
GCF_900002375.1_PBANKA01_protein.faa Plasmodium berghei ANKA 4,896
GCF_900002385.1_PY17X01_protein.faa Plasmodium yoelii 5,926

2. Cleaning and formatting the data to desired specifications

Next step is to clean up this data so that it is easier to handle for both program and for the user. Since these files are identified by accessing id, we will rename them to some easily readable name. We will use the first 4 letters of genus name and first 4 letters of spp name (eg: plaviva for Plasmodium vivax Sal-1).

Files before:

$ ls -1
GCF_000002415.2_ASM241v2_protein.faa
GCF_000002765.4_ASM276v2_protein.faa
GCF_000006355.1_ASM635v1_protein.faa
GCF_000321355.1_PcynB_1.0_protein.faa
GCF_000524495.1_Plas_inui_San_Antonio_1_V1_protein.faa
GCF_000709005.1_Plas_vinc_vinckei_V1_protein.faa
GCF_000956335.1_Plas_frag_nilgiri_V1_protein.faa
GCF_001601855.1_ASM160185v1_protein.faa
GCF_001602025.1_ASM160202v1_protein.faa
GCF_001680005.1_ASM168000v1_protein.faa
GCF_900002335.2_PCHAS01_protein.faa
GCF_900002375.1_PBANKA01_protein.faa
GCF_900002385.1_PY17X01_protein.faa

script to rename files

for faa in *.faa; do
new=$(head -n 1 ${faa} |grep -wEo "Plasmodium .*\b" |cut -f 1-2 -d " "| sed 's/\(\w\w\w\w\)\w*\( \|$\)/\1/g');
mv ${faa} ${new}.fasta;
done

Files after:

Plasberg.fasta
Plaschab.fasta
Plascoat.fasta
Plascyno.fasta
Plasfalc.fasta
Plasfrag.fasta
Plasgabo.fasta
Plasinui.fasta
Plasknow.fasta
Plasreic.fasta
Plasvinc.fasta
Plasviva.fasta
Plasyoel.fasta

Next, we will clean the sequence headers. Although this is not absolutely required for these files, when using custom files it is necessary to keep consistency among all the files being used for the analysis. Here our file fasta header looks like this (all 13 are from NCBI and they are consistent)

Headers before:

$ head -n 1 *.fasta |grep "^>"
>XP_022711843.1 BIR protein, partial [Plasmodium berghei ANKA]
>XP_016652856.1 CIR protein [Plasmodium chabaudi chabaudi]
>XP_019912384.1 Dual specificity phosphatase [Plasmodium coatneyi]
>XP_004220499.1 hypothetical protein PCYB_021010, partial [Plasmodium cynomolgi strain B]
>XP_001347288.1 erythrocyte membrane protein 1, PfEMP1 [Plasmodium falciparum 3D7]
>XP_012333075.1 hypothetical protein AK88_00001, partial [Plasmodium fragile]
>XP_018638642.1 putative EMP1-like protein, partial [Plasmodium gaboni]
>XP_008813840.1 hypothetical protein C922_00001 [Plasmodium inui San Antonio 1]
>XP_002257498.1 SICA antigen (fragment), partial [Plasmodium knowlesi strain H]
>XP_012760262.2 rifin [Plasmodium reichenowi]
>XP_008621913.1 hypothetical protein YYE_00001, partial [Plasmodium vinckei vinckei]
>XP_001608309.1 hypothetical protein [Plasmodium vivax Sal-1]
>XP_022810663.1 YIR protein [Plasmodium yoelii]

Script to modify headers:

for fasta in *.fasta; do
cut -f 1 -d " " $fasta > ${fasta%.*}.temp;
mv ${fasta%.*}.temp $fasta
done

Headers after:

$ head -n 1 *.fasta |grep "^>"
>XP_022711843.1
>XP_016652856.1
>XP_019912384.1
>XP_004220499.1
>XP_001347288.1
>XP_012333075.1
>XP_018638642.1
>XP_008813840.1
>XP_002257498.1
>XP_012760262.2
>XP_008621913.1
>XP_001608309.1

3. Running OrthoMCL program

Since we don’t want to manually install the orthoMCL program and run, We will use the singularity container (made by ISU-HPC) .

module load singularity
singularity pull --name orthomcl.simg shub://ISU-HPC/orthomcl

This will create simg file (containers) for the orthoMCL program

orthomcl.simg

The overview of running OrthoMCL

Fig 2: Overview of OrthoMCL pipeline. Here, all orange boxes will per performed using the orthomcl.simg singularity container and pink boxes in mysql.simg. The BLAST analysis (yellow box) will be done outside the container to speed up the process.

A. Clean and filter sequences

For this, we will create a directory where we store the formatted and filtered sequences. We will use orthomclAdjustFasta and orthomclFilterFasta for these steps.

mkdir -p original complaintFasta
mv *.fasta original/
singularity shell orthomcl.simg
cd complaintFasta
for fasta in ../original/*.fasta; do
orthomclAdjustFasta $(basename ${fasta%.*}) ${fasta} 1
done

The arguments supplied here are the 8 letter prefix for each file, input file and the field in the fasta file that should be used as identifier (in our case it was the first number so it is 1).

For filtering, we will create another directory and save files there:

cd ..
orthomclFilterFasta complaintFasta 10 20

Here, 10 is the minimum length for protein to keep and 20 is the maximum allowed stop codons in the sequences. This command will generate 2 files: goodProteins.fasta, containing all proteins that passed the filtering and poorProteins.fasta, containing all rejects.

We can now exit the singularity image shell and run the next step on a cluster.

exit

B. Find similar sequences using BLAST

Depending the size of sequences, you can either split the goodProteins.fasta file to smaller bits and run the blast step in parallel or run it as single query file against single database (much slower). Here we will just split them to 4 pieces and run them all in a single slurm job.

Load the module and create blast database

module load ncbi-blast
makeblastdb -in goodProteins.fasta -dbtype prot -parse_seqids -out goodProteins.fasta

Split the fasta file to 4 parts

fasta-splitter.pl --n-parts 4 goodProteins.fasta

Generate commands for blast

for split in goodProteins.part-?.fasta; do
echo "blastp -db goodProteins.fasta -query ${split} -outfmt 6 -out ${split}.tsv -num_threads 4";
done > blastp.cmds

Create SLURM submission script and submit the jobs

makeSLURMp.py 4 blastp.cmds
for sub in *.sub; do
sbatch $sub;
done

Once these jobs complete, you can merge the results to a single file

cat goodProteins.part-?.fasta.tsv >> blastresults.tsv

The scripts mentioned here are available on our GitHub repo common_scripts

We will now convert the blast results to format that is needed for uploading to MySQL database

orthomclBlastParser blastresults.tsv ./complaintFasta/ >> similarSequences.txt

This will generate similarSequences.txt file that we will load to MySQL db.

C. Finding Orthologs:

In this section, we will:

i. Set up the MySQL config file so that orthoMCL knows how to access the database for storing/retrieving information ii. Start the MySQL server on the container iii. Setup database/tables for orthoMCL iv. Load the BLAST results and call pairs v. Dump the results (pairs) from the MySQL db as a text file vi. Stop the server vii. Run clustering program (MCL)

i. Setting up config file

This is a simple text file, which provides information for OrthoMCL program about, the database and table name to which you’re loading the blast results, username and password for the MySQL server and some settings (that you can change)

You can create one as follows:

cat > orthomcl.config <<END
dbVendor=mysql
dbConnectString=dbi:mysql:orthomcl:mysql_local_infile=1:localhost:3306
dbLogin=root
dbPassword=my-secret-pw
similarSequencesTable=SimilarSequences
orthologTable=Ortholog
inParalogTable=InParalog
coOrthologTable=CoOrtholog
interTaxonMatchView=InterTaxonMatch
percentMatchCutoff=50
evalueExponentCutoff=-5
oracleIndexTblSpc=NONE
END

or download the one from here:

curl \
https://raw.githubusercontent.com/ISU-HPC/orthomcl/master/orthomcl.config > orthomcl.config

ii. Start MySQL server

We use a singularity instance (new feature in version 2.4) to run the MySQL server service.

module load singularity
singularity pull --name mysql.simg shub://ISU-HPC/mysql

Create local directories for MySQL. These will be bind-mounted into the container and allow other containers to connect to the database via a local socket as well as for the database to be stored on the host filesystem and thus persist between container instances.

mkdir -p ${PWD}/mysql/var/lib/mysql ${PWD}/mysql/run/mysqld

Start the singularity instance for the MySQL server

singularity instance.start --bind ${HOME} \
--bind ${PWD}/mysql/var/lib/mysql/:/var/lib/mysql \
--bind ${PWD}/mysql/run/mysqld:/run/mysqld \
./mysql.simg mysql

Run the container startscript to initialize and start the MySQL server. Note that initialization is only done the first time and the script will automatically skip initialization if it is not needed. This command must be run each time the MySQL server is needed (e.g., each time the container is spun-up to provide the MySQL server).

singularity run instance://mysql

iii. Setup database and tables (schema) for running orthoMCL

create a database named orhtomcl

singularity exec instance://mysql mysqladmin create orthomcl

Since we already have the singularity image for the orthoMCL, we will use the scripts in there to install schema for the database and create all the necessary tables for running next steps.

singularity shell --bind $PWD \
  --bind ${PWD}/mysql/run/mysqld:/run/mysqld \
  ./orthomcl.simg

install schema

 orthomclInstallSchema orthomcl.config

iv. Load BLAST results and call pairs

Next, load the parsed blast results:

orthomclLoadBlast orthomcl.config similarSequences.txt

Call pairs:

orthomclPairs orthomcl.config pairs.log cleanup=no

v. Get the results

orthomclDumpPairsFiles orthomcl.config

The output will be a directory (called pairs) and a file (called mclinput). The pairs directory, will contain three files: orthologs.txt, coorthologs.txt, inparalogs.txt. Each of these files describes pair-wise relationships between proteins. They have three columns: Protein 1, Protein 2 and normalized similarity score between them. The mclinput file contains the identical information as the three files in pairs directory but merged as a single file and in a format accepted by the mcl program.

vi. Stop the MySQL server

Stopping server:

singularity instance.stop mysql

vii. Run the clustering program

MCL program (Dongen 2000) will be used to cluster the pairs extracted in the previous steps to determine ortholog groups.

mcl mclInput --abc -I 1.5 -o groups_1.5.txt

Here, --abc refers to the input format (tab delimited, 3 fields format), -I refers to inflation value and -o refers to output file name. Inflation value will determine how tight the clusters will be. It can range from 1 to 6, but most publications use values between 1.2 -1.5 for detecting orthologous groups. The final step is to name the groups called by mcl program.

orthomclMclToGroups OG1.5_ 1000 < groups_1.5.txt > named_groups_1.5.txt

Here, OG1.5_ is the prefix we use to name the ortholog group, 1000 is the starting number for the ortholog group and last 2 fields are input and output file name respectively.

If you want to test this for other inflation values (ranges from 1.5-6.0), you can create a loop and run this with different I values.

For running it on all possible I values from 1.5-6 wiht increments of 0.5:

for i in $(seq 1.5 0.5 6); do
mcl mclInput --abc -I ${i} -o groups_${i}.txt;
orthomclMclToGroups OG${i}_ 1000 < groups_${i}.txt > named_groups_${i}.txt;
done

4. Formatting and processing results

Next, we will filter the results to select only 1:1 orthologs. For this, we will need a frequency table constructed form the named_groups_1.5.txt file. We can create a simple bash script that counts the number of time each species occurs in each line and create frequency table. Script is available here

CopyNumberGen.sh named_groups_2.5.txt > named_groups_2.5_freq.txt

Since our downstream analysis requires single copy orthologs (SCOs), we will now select the orhtolog groups that have exactly one gene per organism and present in all of them.

ExtractSCOs.sh named_groups_2.5_freq.txt > scos_list_2.5.txt

If you used all possible inflation values, you can run it in a loop:

for i in $(seq 1.5 0.5 6); do
CopyNumberGen.sh named_groups_${i}.txt > named_groups_${i}_freq.txt;
ExtractSCOs.sh named_groups_${i}_freq.txt > scos_list_${i}.txt;
done

By counting the number of lines in scos_list_${i}.txt file, you can count how many single copy orthologs were predicted for various inflation values.

wc -l scos_list_*.txt

in our case, we see that 1.5 value gave us higest number of SCOs

scos_list_1.5.txt	3329
scos_list_2.0.txt	3272
scos_list_2.5.txt	3240
scos_list_3.0.txt	3213
scos_list_3.5.txt	3176
scos_list_4.0.txt	3150
scos_list_4.5.txt	3114
scos_list_5.0.txt	3079
scos_list_5.5.txt	3057
scos_list_6.0.txt	3042

This makes sense, as lower the inflation value, tighter the clusters created by mcl program. We will use the 1.5 value file for the next step.

For each of these SCO ortholog group, we will extract the fasta sequence, and write them to separate file. Eg., OG1297 has 1 gene per oragnism. We will create a file OG1297.fa with 13 sequences each belonging differnt spp. We will create 3,329 such files, required for next step of alignment and tree reconstruction.

Create a list of orthogroups that have SCOs using the file created from the last step

cut -f 1 scos_list_1.5.txt| grep -v "OG_name" | sed 's/$/:/' > scos.ids
grep -Fw -f scos.ids named_groups_1.5.txt > named_groups_1.5_scos.txt

Extract the sequences:

ExtractSeq.sh -o orhtogroups named_groups_1.5_scos.txt  goodProteins.fasta

This will create a folder named orhtogroups, with 3,329 files each containing 13 sequences. This will be the results from this chapter that we will use in the next excercise of tree reconstruction.