Expected number of markers * :

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Results Results

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Results

The utility of a phylogenetic marker can be described by its relative evolutionary rate (RER): faster (respectively slower) evolving markers will be more suitable for lower (respectively deeper) taxonomic levels.

In a first approximation, the total branch length (TBL) of the maximum likelihood (ML) tree is a reasonable descriptor of the evolutionary rate of a given exon / CDS. However, the TBL will preclude fair comparisons among different exons / CDS when the taxon sampling differs: the higher the species number, the longer the TBL.

To circumvent this problem, we use the Super Distance Matrix (SDM) approach [Criscuolo et al. 2006], with a three-step procedure:

1. The ML tree inferred from each of the exons / CDS is converted into a matrix of additive distances by computing the path-length between each pair of species.
2. Each of the matrices is brought closer to the others by a factor (αp), according to the least-squares criterion. This operation is equivalent to multiplying by αp every branch length of the initial trees.
3. Optimal values of the alpha_p parameters are calculated following Criscuolo et al. (2006).
   As αp are inversely proportional to the evolutionary rates, 1/αp values provide a measure of rate heterogeneities among exons / CDS even if the number of taxa differs.
   Here, relative evolutionary rate SDM estimates range from 0.01 to 5 (OrthoMaM mean = 1.2 ; standard-error = 0.6). For example, if exons / CDSs X and Y are respectively characterized by relative rates rX = 0.2 and rY = 2.0, this means that Y is evolving 10 times faster than X.

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Request guidelines: In the [0-5] range of values, type RER < 1 to query rather conserved markers, 1 < RER < 2 for more variable markers, and RER > 2 for fast-evolving ones.

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The substitution rate heterogeneity among sites of the exon / CDS alignment is described by the Γ distribution.

Lower (respectively higher) α values correspond to strong (respectively weak) heterogeneity. If α > 1, the substitution pattern among sites is rather homogeneous.

Request guidelines: In the [0-5] range of values, type alpha < 1 to query markers with stronger among-sites variability, and alpha > 1 for alignments with more evenly distributed variability.

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The percentage of G+C on third codon positions is a descriptor of the degree of base composition heterogeneity.

G+C content is more contrasted on third codon positions than on the whole exon / CDS.

Request guidelines:
In the [20-95] range of values, type %GC3 < 40 to query A+T rich markers,
40 < %GC3 < 60 for more equilibrated markers, and %GC3 > 60 for G+C rich markers.

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Zoonomia genomes have been/will be progressively integrated in NCBI. Since we strongly rely on the NCBI orthology, we prefer to wait until the genomes are annotated by the NCBI consortium to fully integrate them in OrthoMaM.

However here is How to merge zoonomia data with Orthomam, align and infer a tree

#Check that singularity is installed or go to https://docs.sylabs.io/guides/3.0/user-guide/installation.html

#download OMM_MACSE singularity image
singularity pull --arch amd64 library://vranwez/default/omm_macse:v12.01

#download iq-tree
wget --no-check-certificate https://github.com/iqtree/iqtree2/releases/download/v2.2.2.7/iqtree-2.2.2.7-Linux.tar.gz
tar -zxvf iqtree-2.2.2.7-Linux.tar.gz 

zoonomiaTable="overview.table.tsv"
zoonomiaALN="ENST00000678073.ACE2.fasta"
orthomamALN="59272_ACE2_NT_AL.fasta"
mixedALN="ortho_zoonomia_aln.fasta"

#download ace2 from zoonomia 
wget --no-check-certificate https://genome.senckenberg.de/download/TOGA/human_hg38_reference/MultipleCodonAlignments/$zoonomiaALN.gz

#download orthomam NT alignment
wget --no-check-certificate https://orthomam.mbb.cnrs.fr/orthomam_v12/cds/omm_filtered_NT_CDS/$orthomamALN.zip

#merge orthomam and zoonomia files
gunzip $zoonomiaALN.gz

wget --no-check-certificate https://genome.senckenberg.de/download/TOGA/human_hg38_reference/$zoonomiaTable

sed -i -e 's/Microcebus sp. 3 GT-2019/Microcebus sp/' $zoonomiaTable
sed -i -e 's/REFERENCE/Homo_sapiens/g' $zoonomiaALN

unzip -j $orthomamALN.zip

# A more explicit and readable version
sed -i -e 's/-//g' $zoonomiaALN
sed -i -e 's/REFERENCE/Homo_sapiens/g' $zoonomiaALN
while IFS=$'\t' read -r Species  CommonName  ID  Lineage    Assembly    NCBI    contigN50   scaffoldN50 ; do
   sed -i -e "s/^>vs_$Assembly.*/>$Species/g" $zoonomiaALN
done < $zoonomiaTable   

sed -i -e 's/ /_/g' $zoonomiaALN

cat $zoonomiaALN $orthomamALN > ${orthomamALN}_all
sed -i 's/-//g' ${orthomamALN}_all

unset species; unset species_sequence
declare -A  species_sequence
declare species

while read -r line   ; do
    if [[ $line == ">"* ]] ;
    then
        species=${line#*">"}
    else 
        if [[ ${#line} -gt ${#species_sequence["$species"]} ]] ;
        then
            species_sequence["$species"]=`echo $line` #we take the longest transcript 
        fi  
    fi
done <  ${orthomamALN}_all  

echo -n > $mixedALN
for KEY in "${!species_sequence[@]}"; do
  echo ">$KEY" >> $mixedALN
  echo "${species_sequence[$KEY]}" >> $mixedALN
done

rm ${orthomamALN}_all

# fast one pass alternative :  but less readable
# awk 'BEGIN {FS="\t"}
# {
#   if (NR == FNR) {#we are reading overview.table.tsv so create a dict mapping Assembly names to species names
#     gsub(/ /,"_",$1);    species[">vs_"$5] = $1 #replace space in species names with _
#   }  
#   else {
#     if (index($0, ">") == 1) #seq ident
#     { 
#       split($0, ID, " ")  
#       assembly=ID[1] #keep only first part of ident which is assembly name in zoonomia
#       if (assembly in species) cur_species = ">"species[assembly] #replace assembly name with species name
#       else cur_species = assembly #probable missing assembly ident or orthomam ident
#     }
#     else {gsub(/-/,"",$0);if(length($0)>length(species_sequence[cur_species])) species_sequence[cur_species]=$0}  # remove gaps from sequence and keep the longest
#  }
# }
# END {for (sp in species_sequence) {print sp"\n"species_sequence[sp] } } 
# ' $zoonomiaTable $zoonomiaALN $orthomamALN > ${mixedALN}

#Run macse

./omm_macse_v12.01.sif --in_seq_file ENST00000678073.59272_NT_AL.fasta \
 --out_dir ALIGN_ACE2_zoonomia_orthomam --out_file_prefix ACE_zoonomia_orthomam --genetic_code_number 1 --min_percent_NT_at_ends 0.2 \ 
 --no_filtering --no_FS_detection

#Run IQ-tree  to reconstruct a maximum-likelihood tree using ModelFinder
iqtree-2.2.2.7-Linux/bin/iqtree2 -s ALIGN_ACE2_zoonomia_orthomam/ACE_zoonomia_orthomam_final_align_NT.aln -m MFP