Table of Contents

• Prepare data
• Installation
• Run OrthoFinder for orthology
• Run WGDI for synteny
• Run SOI for orthologous synteny and species tree

Prepare data

Download and pre-process the example data:

# git-lfs has been installed
git lfs clone https://github.com/zhangrengang/evolution_example
cd evolution_example

chmod +x src/*
find . -name "*gz" | xargs gunzip
cat OrthoFinder/*fasta > pep.faa
cat CDS/* > cds.fa
cat wgdi/*gff > all_species_gene.gff

If it is hard to download from GitHub, altenatively you can try to download from BaiduYun. Now, the directory structure like this:

$ tree
├── all_species_gene.gff        # all GFF
├── cds.fa                      # all CDS sequences
├── pep.faa                     # all protein sequences
├── species.design              # speceis list
├── OrthoFinder                 # input for OrthoFinder
│   ├── Angelica_sinensis.fasta
│   ├── Apium_graveolens.fasta
│   ├── ......
└── wgdi                        # input for WGDI
    ├── Angelica_sinensis-Angelica_sinensis.blast
    ├── Angelica_sinensis-Angelica_sinensis.conf
    ├── Angelica_sinensis-Angelica_sinensis.ctl   # for `soi dotplot`
    ├── Angelica_sinensis.gff
    ├── Angelica_sinensis.lens
    ├── ......
 ......

Note: the GENE ID is needed to label with SPECIES ID (e.g. Angelica_sinensis|AS01G00001) for uniqueness and compatibility (legacy from OrthoMCL). The CHROMosome ID should also be unique (e.g. As1, As2) to avoid conflicts (legacy from MCscanX). The IDs can be easily labeled for your own data; for example:

SP=Angelica_sinensis
# using OrthoMCL command for fasta files:
orthomclAdjustFasta $SP $SP.pep 1
# or using sed:
sed 's/>/>'$SP'|/' $SP.pep > $SP.fasta  # always using the separator '|'

SP2=As
# using awk for MCscanX/WGDI gff files:
awk -v sp=$SP -v OFS="\t" '{$2=sp"|"$2;print $0}' $SP.gff0 | perl -pe 's/^\D+/'$SP2'/' > $SP.gff

SP and SP2 can be the same.

Installation

If you have installed OrthoIndex, all the commands used in this pipeline should have been installed.

Run OrthoFinder for orthology

To infer 'orthology' using OrthoFinder2:

orthofinder -f OrthoFinder/ -M msa -t 60

orthofinder can by replaced by Proteinortho6, Broccoli and SonicParanoid2.

Run WGDI for synteny

To detect 'synteny' by WGDI, with visualization by SOI :

cd wgdi

../src/comb2 `cat ../species.design` | while read LINE
do
    arr=($LINE)
    SP1=${arr[0]}
    SP2=${arr[1]}
    prefix=$SP1-$SP2
    conf=$prefix.conf

    # blast
    diamond blastp -q ../OrthoFinder/$SP1.pep -d ../OrthoFinder/$SP2.pep -o $prefix.blast --more-sensitive -p 10 --quiet -e 0.001

    # call synteny
    wgdi -icl $conf

    # dot plot colored by Orthology Index
    soi dotplot -s $prefix.collinearity \
        -g ../all_species_gene.gff -c $prefix.ctl  \
        --xlabel $SP1 --ylabel $SP2 \
        --ks-hist --max-ks 1 -o $prefix.io    \
        --plot-ploidy --gene-axis --number-plots \
        --ofdir ../OrthoFinder/OrthoFinder/Results_*/ \
        --of-color

    # to show only orthology
    soi dotplot -s $prefix.collinearity \
        -g ../all_species_gene.gff -c $prefix.ctl  \
        --xlabel $SP1 --ylabel $SP2 \
        --ks-hist --max-ks 1 -o $prefix.io    \
        --plot-ploidy --gene-axis --number-plots \
        --ofdir ../OrthoFinder/OrthoFinder/Results_*/ \
        --of-color --of-ratio 0.6

done

cd ..

If you also need Ks-based visualization:

cd wgdi

../src/comb2 `cat ../species.design` | while read LINE
do
    arr=($LINE)
    SP1=${arr[0]}
    SP2=${arr[1]}
    prefix=$SP1-$SP2
    conf=$prefix.conf

    # calculate Ks
    wgdi -ks $conf

    # dot plot colored by Ks
    soi dotplot -s $prefix.collinearity \
        -g ../all_species_gene.gff -c $prefix.ctl  \
        --kaks $prefix.collinearity.ks \
        --xlabel $SP1 --ylabel $SP2 \
        --ks-hist --max-ks 1.5 -o $prefix     \
        --plot-ploidy --gene-axis --number-plots

    # to show only orthology
    soi dotplot -s $prefix.collinearity \
        -g ../all_species_gene.gff -c $prefix.ctl  \
        --kaks $prefix.collinearity.ks \
        --xlabel $SP1 --ylabel $SP2 \
        --ks-hist --max-ks 1.5 -o $prefix     \
        --plot-ploidy --gene-axis --number-plots \
        --ofdir ../OrthoFinder/OrthoFinder/Results_*/ \
        --of-ratio 0.6       # filtering by OI

done

cd ..

wgdi can be replaced by JCVI and MCscanX.

Run SOI for orthologous synteny and species tree

To cluster syntenic orthogroups (SOGs) and construct phylogenomic analyses:

cd phylogenomics

# to filter collinearity
soi filter -s ../wgdi/*.collinearity -o ../OrthoFinder/OrthoFinder/Results_*/ -c 0.6 > collinearity.ortho

# to cluster SOGs excluding outgroups that do not share the lineage-specific WGD
soi cluster -s collinearity.ortho -outgroup Lonicera_japonica Ilex_polyneura Vitis_vinifera -prefix cluster
# to add outgroups
soi outgroup -s collinearity.ortho -og cluster.mcl -outgroup Lonicera_japonica Ilex_polyneura Vitis_vinifera > cluster.mcl.plus

# to build multi-copy or single-copy gene trees
soi phylo -og cluster.mcl.plus -pep ../pep.faa -cds ../cds.fa -both -pre sog -mm 0.4 -p 80 -tmp tmp.mc.0.4
soi phylo -og cluster.mcl.plus -pep ../pep.faa -cds ../cds.fa -both -pre sog -mm 0.2 -p 80 -tmp tmp.sc.0.2 -sc -concat -trimal_opts " -gappyout" -iqtree_opts " -B 1000"

# to infer coalescent‐based species tree
astral-pro --root Vitis_vinifera sog.mc.cds.mm0.4.genetrees > sog.mc.cds.mm0.4.genetrees.astral
astral-hybrid --root Vitis_vinifera sog.sc.cds.mm0.2.genetrees > sog.sc.cds.mm0.2.genetrees.astral

# to infer concatenation‐based species tree
iqtree2 -s sog.sc.cds.mm0.2.concat.aln -T 60 -B 1000 -mset GTR -o Vitis_vinifera

Note: although we set a unified cutoff (0.6) in the pipeline, users should manually check the resulted dot plots for confirmation, and some extremely complex cases showing unexpected patterns need to be investigated on a case-by-case basis.