Mirror sites for analyses
ORTHOSCOPE (Inoue and Satoh 2019) is a web tool to identify orthogroup members (orthologs and paralogs, see below) of a specific protein-coding gene of animals and plants. By uploading gene sequences of interest and by selecting species genomes from >600 animals/plants, 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 an orthogroup.
ORTHOSCOPE works only for a specific molecule and does not allow genome-scale analyses. Recently I developed the downloaded version, ORTHOSCOPE* (star). This analytic pipeline accommodates genome-wide data of protein-coding genes and infers genome-scale events.
Japanese instruction (日本語の説明):
Togo TV (日本語の動画):
Genome wide analayses:
A set of genes descended from a single gene in the last common ancestor of all the species being considered (Emms and Kelly 2015).
Broadly Accepeted Nodes Used in Focal Analyses
|Focal analysis||Broadly Accepeted Nodes|
|Actinopterygii||Teleostomi, Gnathostomata, or Vertebrata|
|Mammalia||Amniota, Tetrapoda, or Sarcopterygii|
|Vertebrata||Olfactore, Chordata, or Deuterostomia|
|Deuterostomia||Nephrozoa or Bilateria|
|Protostomia||Nephrozoa or Bilateria|
|Acropora||Scleractinia, Anthozoa, or Cnidaria|
Use of Query Sequences in Gene Tree Estimation
Redundant Blast hits are deleted
Queries are added or replaced
Ishikawa et al (2019)
Ishikawa, A, et al. 2019. A key metabolic gene for recurrent freshwater colonization and radiation in fishes. Science, 364: 886-9. Link.
Inoue et al (2019)
Inoue J, Nakashima K, and 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
For queries, CesA gene sequences were separated into 2 parts: CesA (before TM7) and GH6 (after TM7]) domains.
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. 36:621–631. Link.
Downloading query sequences from NCBI/Ensembl
Collecting Query Sequences from an Assemble Database (Vertebrate ALDH1A and Actinopterygin PLCB1)
- Download Coregonus lavaretus TSA file (GFIG00000000.1) form NCBI.
- Translate raw sequences into amino acid and coding sequences using TransDecoder.
./TransDecoder.LongOrfs -t GFIG01.1.fsa_nt
- Make blast databases using BLAST+.
makeblastdb -in longest_orfs.pep -dbtype prot -parse_seqids makeblastdb -in longest_orfs.cds -dbtype nucl -parse_seqids
- BLASTP seaech against amino acid database.
blastp -query query.txt -db longest_orfs.pep -num_alignments 10 -evalue 1e-12 -out 010_out.txt
- 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
Case 1: Query seqeunce is present in the ORTHOSCOPE database
Case 2: Query seqeunce is not present in the ORTHOSCOPE database
Rooting Selection from Blast Hits
Species Tree Hypothesis
See our Species_tree page.
Aligned Site Rate
Rearrangement BS value threshold
Genome Taxon Sampling
To reduce computational burden, "Number of species" is restricted.
For example, if a user selected "Number of hits to report per genome" as 3, "Number of species" should be less than 50 spp.
|Number of hits to report per genome||Number of species|
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: DeuterostomeBra_2ndAnalysis.zip.
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: https://github.com/stamatak/standard-RAxML
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:
trimAl v1.2 (Official release):
Available here: http://trimal.cgenomics.org/downloads.
Cd to trimAl/source, type make, and copy the executable.
make cp trimal ~/bin
Available here: http://www.bork.embl.de/pal2nal/#Download.
Change the permission of perl script and copy it.
chmod 755 pal2nal.pl cp pal2nal.pl ~/bin
Ape in R:
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:
Decompress DeuterostomeBra_2ndAnalysis.zip. Open DeuterostomeBra_2ndAnalysis file and decompress 100_2ndTree.tar.gz file.
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.
- Cd into 100_2ndTree directory.
- Run the pipeline.
- ML tree is saved in 200_RAxMLtree_Exc3rd.pdf automatically.
Duplicated Node Estimation
Using Notung, duplicated nodes can be identified. Here, we will analyze the gene tree of orthogroup members.
- Double click the downloaded .jar file (here, Notung-2.9.jar).
- Save the species tree (newick format) as a new file (here, speciesTree.tre), from 000_summary.txt file.
- Open the species tree file, speciesTree.tre (File > Open Gene Tree), from Notung.
- Open the gene tree file, RAxML_bootstrap.txt (File > Open Gene Tree).
- Set "Edge Weight THreshold" (here 70) from “Edit Values button“. This value corresponds to “Rearrangement BS value threshold” in ORTHOSCOPE.
- From "Rearrange" tab in the bottum, select "Prefix of the general label".
- Push "Reconcile" button.
- Duplicated nodes are shown with "D".
|Supported||Supported||11.0 or later||Not supported|
Gene Model Databases of 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.