Finding Orthologs and Paralogs

Orthologs

Gene families and Orthology

Problem: How to find “same” genes across multiple species.

Genes can duplicate (Paralogs) and can be identical due to descent (Ortholog)

orthologs

Methods

  • BLAST: 1 way BLAST (Gene A in Species X, what is best hit in Species Y)
  • BLAST: reciprocal BLAST

diagramorth

Trees can help resolve relationships

Best hits can sometimes be wrong (B) though it can be resolved with phylogenetics.

RIO

Reciprocal Searches

  • Bi-directional or Reciprocal BLAST

BRH

Implement Bidirectional

Method to find best top hit in one direction and the reverse.

Let’s walk through the code

Will write this in Python in Class

Clustering

  • Lumping genes together based on similarity linkage
  • Single-linkage means if there is a link between A-B then they are in a cluster

SingleLinkage

Code up single-linkage

Let’s look at some code.

Will write this in Python in Class

Issues

orthologsloss

Tools to go after Orthologous and Paralogous Sequences

Steps to build orthologs on cluster

We will take 3 datasets of annotated Cyanobacteria, download and run analysis to generate Ortholog table.

#!/bin/bash -l
#SBATCH -p short -N 1 -n 1 -c 8 --mem 8G --time 2:00:00
#SBATCH -J orthofinder
#SBATCH -o logs/%x.%j.log
module load ncbi-blast
module load orthofinder

set -euo pipefail
CPU=${SLURM_CPUS_PER_TASK:-1}

mkdir -p cyanobacteria
cd cyanobacteria
curl -L -O ftp://ftp.ensemblgenomes.org/pub/bacteria/release-45/fasta/bacteria_10_collection/oscillatoriales_cyanobacterium_jsc_12/pep/Oscillatoriales_cyanobacterium_jsc_12.ASM30994v1.pep.all.fa.gz
curl -L -O ftp://ftp.ensemblgenomes.org/pub/bacteria/release-45/fasta/bacteria_0_collection/nostoc_punctiforme_pcc_73102/pep/Nostoc_punctiforme_pcc_73102.ASM2002v1.pep.all.fa.gz
curl -L -O ftp://ftp.ensemblgenomes.org/pub/bacteria/release-45/fasta/bacteria_4_collection/cyanobacterium_aponinum_pcc_10605/pep/Cyanobacterium_aponinum_pcc_10605.ASM31767v1.pep.all.fa.gz

# uncompress files and name them all *.fasta
for file in *.fa.gz
do
 m=$(basename $file .pep.all.fa.gz)
 pigz -dc $file > $m.fasta
done

cd ..

# -t: threads for the all-vs-all sequence searches, -a: threads for the tree/analysis steps
orthofinder -t $CPU -a $CPU -f cyanobacteria

Run mkdir -p logs before you sbatch it. See UNIX IV for how to choose -c, --mem and --time.

Ortholog results

The output file by default will be the date of the analysis. Opening the file cyanobacteria/Results_XXX/Orthogroups.txt but I made a folder in the examples you can look over. Here’s one table

Format 

GroupName\tSp1_Gene1, Sp1_Gene2\tSp2_Gene1, Sp2_Gene2\tSp3_Gene1, Sp3_Gene2

	Cyanobacterium_aponinum_pcc_10605.ASM31767v1	Nostoc_punctiforme_pcc_73102.ASM2002v1	Oscillatoriales_cyanobacterium_jsc_12.ASM30994v1
OG0000000			EKQ66605, EKQ66611, EKQ66662, EKQ66782, EKQ66954, EKQ66984, EKQ67084, EKQ67085, EKQ67278, EKQ67284, EKQ67330, EKQ67344, EKQ67361, EKQ67366, EKQ67368, EKQ67427,
 EKQ67433, EKQ67590, EKQ67680, EKQ67799, EKQ67807, EKQ67983, EKQ68026, EKQ68032, EKQ68054, EKQ68173, EKQ68296, EKQ68354, EKQ68495, EKQ68500, EKQ68912, EKQ69012, EKQ69027, EKQ69073, EKQ69085,
EKQ69279, EKQ69300, EKQ69345, EKQ69368, EKQ69506, EKQ69549, EKQ69629, EKQ69630, EKQ69655, EKQ69739, EKQ69760, EKQ69872, EKQ69984, EKQ70005, EKQ70023, EKQ70046, EKQ70130, EKQ70179, EKQ70602, E
KQ70786, EKQ70840, EKQ70870, EKQ70894, EKQ71088, EKQ71090, EKQ71265, EKQ71335
OG0000001	AFZ52442, AFZ54265, AFZ54640	ACC78968, ACC78978, ACC79054, ACC79090, ACC79138, ACC79182, ACC79941, ACC80158, ACC80645, ACC81000, ACC81451, ACC81520, ACC81612, ACC81781, ACC
81797, ACC82091, ACC82628, ACC82978, ACC83035, ACC83215, ACC83711, ACC84528, ACC84844, ACC84851, ACC85118, ACC85266, ACC85421	EKQ66618, EKQ66620, EKQ66621, EKQ67508, EKQ68220, EKQ69508, EKQ
69971, EKQ69995, EKQ70003, EKQ70556, EKQ70833, EKQ71286
OG0000002	AFZ55137	ACC79344, ACC80485, ACC80595, ACC82143, ACC82836, ACC82962, ACC83845, ACC83999, ACC84046, ACC84047, ACC84257, ACC84700, ACC84955, ACC84966, ACC84970, ACC84971,
 ACC84972, ACC84974, ACC84981, ACC84982, ACC84983, ACC85032	EKQ66950, EKQ67597, EKQ67615, EKQ68095, EKQ68496, EKQ68499, EKQ69362, EKQ69910, EKQ69959
OG0000003	AFZ53198	ACC78875, ACC78976, ACC79256, ACC79524, ACC79759, ACC80145, ACC80528, ACC80591, ACC80631, ACC80670, ACC80818, ACC81317, ACC81502, ACC82310, ACC82674, ACC82718,
 ACC82769, ACC83025, ACC83081, ACC83457, ACC83602, ACC83721, ACC83749, ACC84422, ACC85331	EKQ69908
OG0000004		ACC80422, ACC80525, ACC80662, ACC80851, ACC80857, ACC80914, ACC81440, ACC81547, ACC81570, ACC81705, ACC81945, ACC81949, ACC81968, ACC81987, ACC82586, ACC83096, ACC8342
6, ACC83981, ACC84622, ACC84732, ACC85457	EKQ66830, EKQ66911, EKQ67039, EKQ67311, EKQ69997, EKQ70735
OG0000005	AFZ52318, AFZ52611, AFZ52613, AFZ52925, AFZ52973, AFZ53626, AFZ53840, AFZ53841, AFZ53849, AFZ53949, AFZ54004, AFZ54551, AFZ54904, AFZ55229	ACC80197, ACC81960, ACC82068, A
CC82559, ACC83603, ACC83674, ACC85005, ACC85009	EKQ67574, EKQ67809, EKQ69976
OG0000006	AFZ52319, AFZ53394, AFZ54017, AFZ54472	ACC79360, ACC79745, ACC79853, ACC80832, ACC80867, ACC81560, ACC82363, ACC82406, ACC82436, ACC82585, ACC84067, ACC84385	EKQ67267, EKQ67
478, EKQ67551, EKQ67724, EKQ67810, EKQ68266
OG0000007	AFZ53704, AFZ54461, AFZ54462	ACC79786, ACC80242, ACC80282, ACC80538, ACC80768, ACC81387, ACC82067, ACC83125, ACC83317, ACC83447, ACC84291, ACC84302, ACC85008	EKQ6767
2, EKQ68369, EKQ70142, EKQ70145, EKQ71300

The tool also generates summary statistics we can look through.

Could write a script to turn this into a table or use the summary count table provided.

ORTHOLOG_GRP	SP1   SP2    SP3
ORTHO_0001         10     5   0
ORTHO_0002          1     1   0