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ORTHOSCOPE ( (Inoue and Satoh 2019) is a web tool to identify orthologs of a specific protein-coding gene of bilaterians (and cnidarians). By uploading gene sequences of interest and by selecting species genomes from >450 bilaterians, users can infer their functions and copy numbers, according to results reported by ORTHOSCOPE in the form of gene trees.

By using sequences collected by the BLAST search, ORTHOSCOPE estimates the gene tree, compares it with the species tree, and identifies a orthogroup (see below).

Japanese instruction (日本語の説明):

Web sites

Mirror AORI (from 28 July 2020)

Mirror SAKURA VPS (from 29 December 2020)


A set of genes descended from a single gene in the last common ancestor of all the species being considered (Emms and Kelly 2015).



Flow Chart


BLAST 2.7.1+
MAFFT v7.356b
trimAl 1.2rev59
ape in R, Version5.0
FastME 2.0 for amino acid analyses

Use of Query Sequences in Gene Tree Estimation

Redundant Blast hits are deleted


Queries are added or replaced


Example Data

Inoue et al (2019)

Inoue J, Nakashima K, and Satoh N. ORTHOSCOPE analysis reveals the presence of the cellulose synthase gene in all tunicate genomes but not in other animal genomes. Genes. 10: 294.

Queries. These sequences were used for "Tree Search Only" mode.
In this paper, maximum likelihood trees were estimated according to the process described in "Tree Estimation of Orthogroup Members (with Additional Sequences)". See below.

Inoue and Satoh (2019)

Inoue J. and Satoh N. 2019. ORTHOSCOPE: an automatic web tool of analytical pipeline for ortholog identification using a species tree. MBE in press.

Actinopterygii Vertebrata Deuterostomia Protostomia
PLCB1* ALDH1A* Brachyury Brachyury
Queries Queries Queries Queries
Result Result Result Result

Downloading query sequences from NCBI/Ensembl

From NCBI or Ensembl, query sequences can be downloaded.
For coding sequneces, please select CDS as follows.


Collecting Query Sequences from an Assemble Database (Vertebrate ALDH1A and Actinopterygin PLCB1)

  1. Download Coregonus lavaretus TSA file (GFIG00000000.1) form NCBI.
  2. Translate raw sequences into amino acid and coding sequences using TransDecoder.
./TransDecoder.LongOrfs -t GFIG01.1.fsa_nt
  1. Make blast databases using BLAST+.
makeblastdb -in longest_orfs.pep -dbtype prot -parse_seqids 
makeblastdb -in longest_orfs.cds -dbtype nucl -parse_seqids
  1. BLASTP seaech against amino acid database.
blastp -query query.txt -db longest_orfs.pep -num_alignments 10 -evalue 1e-12 -out 010_out.txt
  1. Retrieve blast top hit sequences from coding sequence file using sequence id.
blastdbcmd -db longest_orfs.cds -dbtype nucl -entry_batch queryIDs.txt -out 020_out.txt

Focal Group

analisis group

Upload Files

Coding sequence

file format

Case 1: Query seqeunce is present in the ORTHOSCOPE database

registered sequence search

Case 2: Query seqeunce is not present in the ORTHOSCOPE database

unregistered sequence search

Rooting Selection from Blast Hits


Species Tree Hypothesis


See our Species_tree page.

Sequence Collection

sequence collection

Aligned Site Rate

sequence alignment

Tree Search


codon mode

Rearrangement BS value threshold

branch rearrangement

NJ analysis is conducted using the software package Ape in R (coding) and FastME (amino acid). Rearrangement analysis is done using a method implemented in NOTUNG.

Genome Taxon Sampling

Feasibility of completion

Number of hits to report per genome Number of species
3 <50
5 <40
10 <30

Tree Estimation of Orthogroup Members (with Additional Sequences)

By using sequences of ORTHOSCOPE results, the analysis can be done on your own computer.
I made an analysis pipeline for this 2nd step. The script is specialized for a Macintosh use with Python 3. Windows users need some modifications.
Analysis pipeline with example data:

Installing Dependencies

Estimation of the 2nd tree by the downloaded pipeline requires some dependencies to be installed and in the system path in your computer.


Available here:

Download the the latest release and extract it. Cd into the extracted directry (e.g., standard-RAxML-8.2.12), compile the PThreads version, and copy the executable to a directory in your system path, e.g.:

cd standard-RAxML-8.2.12
make -f Makefile.SSE3.PTHREADS.gcc
cp raxmlHPC-PTHREADS-SSE3 ~/bin

Add the address to your PATH. For example:

export PATH=$PATH:~/bin

Mafft v7.407:

Available here:
After compilation, set your PATH following this site.

trimAl v1.2 (Official release):

Available here:
Cd to trimAl/source, type make, and copy the executable.

cp trimal ~/bin


Available here:
Change the permission of perl script and copy it.

chmod 755
cp ~/bin

Ape in R:

R (3.5.2) is available from here.
By installing R, rscript will be installed automatically.
APE in R can be installed from the R console as follows:


Tree Estimation

Using the downloaded pipeline, the 2nd gene trees will be estimated as follows:

  • Based on the estimated rearranged NJ tree, users should select coding sequences of orthogroup and outgroups manually. Then the pipeline can start subsequent analyese.
  • Selected sequences are aligned using MAFFT (Katoh et al. 2005).
  • Multiple sequence alignments are trimmed by removing poorly aligned regions using TRIMAL 1.2 (Capella-Gutierrez et al. 2009) with the option “gappyout.”
  • Corresponding cDNA sequences are forced onto the amino acid alignment using PAL2NAL (Suyama et al. 2006) to generate nucleotide alignments.
  • Phylogenetic analysis is performed with RAxML 8.2.4 (Stamatakis et al. 2014), which invokes a rapid bootstrap analysis and searches for the best-scoring ML tree with the GTRGAMMA (Yang 1994a, 1994b) or GTRCAT model.

The actual rocess is as follows:

  1. Decompress Open DeuterostomeBra_2ndAnalysis file and decompress 100_2ndTree.tar.gz file.

  2. Select an appropriate outgroup and orthogroup members and save 010_candidates_nucl.txt file. The outgroup sequence should be placed at the top of alignment. Additional sequences can be included.

query sequences

  1. Cd into 100_2ndTree directory.
  2. Run the pipeline.
  1. ML tree is saved in 200_RAxMLtree_Exc3rd.pdf automatically.

ML tree

Duplicated Node Estimation

Using Notung, duplicated nodes can be identified. Here, we will analyze the gene tree of orthogroup members.

  1. Double click the downloaded .jar file (here, Notung-2.9.jar).
  2. Save the species tree (newick format) as a new file (here, speciesTree.tre), from 000_summary.txt file.
  3. Open the species tree file, speciesTree.tre (File > Open Gene Tree), from Notung.
  4. Open the gene tree file, RAxML_bootstrap.txt (File > Open Gene Tree).
  5. Set "Edge Weight THreshold" (here 70) from “Edit Values button“. This value corresponds to “Rearrangement BS value threshold” in ORTHOSCOPE.
  6. From "Rearrange" tab in the bottum, select "Prefix of the general label".
  7. Push "Reconcile" button.
  8. Duplicated nodes are shown with "D".

Rearranged tree

Supported Browsers

Chrome Firefox Safari IE
Supported Supported 11.0 or later Not supported


Date Version Revision
29 Dec. 2020 Version 1.5.0 Text areas were introduced for sequence uploading. In conjunction with the renewal, the file uploading system was closed.
24 Dec. 2020 Gene model data were newly added for 4 snakes (Pantherophis guttatus, Thamnophis elegans, Naja naja, and Laticauda laticaudata).
6 Dec. 2020 Version 1.2.2 Gene model data were newly added for 3 sharkes (Scyliorhinus torazame, Chiloscyllium punctatum, and Rhincodon typus), human (Homo sapiens Ens102), and chicken (Gallus gallus Ens102).
6 Sep. 2020 Version 1.2.1 Gene model data were newly added for an echinoderm (Anneissia japonica) and replaced with TSA data for an acoela (Hofstenia-miamia).
30 Aug. 2020 Data of Sterlet (Acipenser ruthenus) and European eel (Anguilla anguilla) were newly added.
1 Jun. 2020 Version 1.2.0( Released. A focal group, Acropora, was newly added.
1 Jun. 2020 Version 1.1.0 Released. Data of Amblyraja radiata (Thorny skate) was newly added.
14 Jan. 2020 The batch uploading was implemented for taxon sampling.
6 Nov. 2019 ORTHOSCOPE-Mammalia was newly created and data of 46 mammals were newly added.
2 Oct. 2019 Data of Pacific white shrimp (Penaeus vannamei) were newly added.
5 Sep. 2019 Data of 2 molluscs (Octopus vulgaris, Pomacea canaliculata) were newly added.
21 Aug. 2019 Column of Seqs (# of sequence in each gene model) was added.
21 Aug. 2019 Data of 6 actinopterygians (Erpetoichthys calabaricus, Denticeps clupeoides, Carassius auratus, Electrophorus electricus,Tachysurus fulvidraco, Pangasianodon hypophthalmus), 2 amphibians (Rhinatrema bivittatum, Microcaecilia unicolor), and 3 lepidosaurians (Notechis scutatus, Podarcis muralis, Pseudonaja textilis) were newly added.
19 Apr. 2019 Nagative branch lengths are replaced with 0 in the tree drawing (R script). Gene_tree$edge.length[Gene_tree$edge.length<0]<-0
25 Jan. 2019 Version 1.0.2 Released. For Inoue et al. 2019, Data of Archaea, Plants, Bacteria, and Urochordata were newly added.
21 Dec. 2018 Version 1.0.1 Released. In the rearranged gene tree, nodes identified as speciation events were marked with "D".
18 Dec. 2018 Version 1.0.1.beta Xenacoelomorph, platyhelminth, priapulid, avian data were newly added.
10 July 2018 Version 1.0 Published in Inoue and Satoh (2018).

Gene Model Databases Used in ORTHOSCOPE

ORTHOSCOPE employs a genome-scale protein-coding gene database (coding and amino acid sequence datasets) constructed for each species. In order to count numbers of orthologs in each species, only the longest sequence is used when transcript variants exist for single locus.

29 Dec. 2020
From ver. 1.5.0, each gene model can be downloaded by clicking p (amino acid sequence) or n (coding sequence) in at the right of each species line.

6 Oct. 2019
Gene model databases (fasta files of amino acid and coding sequences) can be downloaded from zenodo (10.5281/zenodo.2553737).

Papers using ORTHOSCOPE

Ishikawa, A, Kabeya, N, Ikeya, K, Kakioka, R, Cech, JN, Osada, N, Leal, MC, Inoue J, Kume, M, Toyoda, A, Tezuka, A, Nagano, AJ, Yamasaki, YY, Suzuki, Y, Kokita, T, Takahashi, H, Lucek, K, Marques, D, Takehana, Y, Naruse, K, Mori, S, Monroig, O, Ladd, N, Schubert, C, Matthews, B, Peichel, CL, Seehausen, O, Yoshizaki, G, Kitano J. 2019. A key metabolic gene for recurrent freshwater colonization and radiation in fishes. Science, 364: 886-9. Link.

By counting the number of gene copies in 48 actinopterygians, ORTHOSCOPE found that Fads2 gene (involved in fatty acid desaturation) was duplicated in freshwater species.

Inoue J, Nakashima K, Satoh, N. 2019. ORTHOSCOPE analysis reveals the presence of the cellulose synthase gene in all tunicate genomes but not in other animal genomes. Genes. 10: 294. Link

By showing the absence of CesA gene in protostomes and basal deuterostomes, ORTHOSCOPE confirmed that the prokaryotic cellulose synthase gene (CesA) was horizontally transferred into the genome of a tunicate ancestor.

Shiraishi A, Okuda T, Miyasaka N, Osugi T, Okuno Y, Inoue J, and Satake H. 2019. Repertoires of G protein-coupled receptors for Ciona-specific neuropeptides. Proceedings of the National Academy of Sciences of the United States of America. 116: 7847-7856. Link

This paper identified multiple G protein-coupled receptors (GPCRs) for species-specific neuropeptides of Ciona intestinalis. By reconstructing gene trees, ORTHOSCOPE showed that these GPCRs are evolutionarily unrelated to any other known peptide GPCRs.

Yasuoka Y, Matsumoto M, Yagi K, Okazaki Y. 2019. Evolutionary History of GLIS Genes Illuminates Their Roles in Cell Reprograming and Ciliogenesis. Molecular Biology and Evolution 37:100-109.

The GLIS family transcription factors, GLIS1 and GLIS3, potentiate generation of induced pluripotent stem cells (iPSCs), although another GLIS family member, GLIS2, suppresses cell reprograming. Using ORTHOSCOPE, Yasuoka et al. showed that GLIS1 and GLIS3 originated during vertebrate whole genome duplication, whereas GLIS2 is a sister group to GLIS1/3. This study clearly indicates that as the first step, future reprograming studies should focus on GLIS1/3 rather than on GLIS2.

Inoue J, Satoh N. 2018. Deuterostome genomics: Lineage-specific protein expansions that enabled chordate muscle evolution. Molecular Biology and Evolution. 35(4):914-924. Link

The pipeline implemented in ORTHOSCOPE was used to evaluate the presence or absence of genes coding for structural-muscle proteins.

Inoue J, Yasuoka Y, Takahashi H, Satoh N. 2017. The chordate ancestor possessed a single copy of the Brachyury gene for notochord acquisition. Zoological Letters. 3: 4. Link

The pieline implemented in ORTHOSCOPE was used to count the number of Brachury gene in 5 deuterostome lineages.


Inoue J. and Satoh N. 2019. ORTHOSCOPE: An automatic web tool for phylogenetically inferring bilaterian orthogroups with user-selected taxa. Molecular Biology and Evolution, 36, 621–631. Link.




An automatic web tool for phylogenetic inference of bilaterian orthogroups under purposeful taxonomic sampling



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