For identifying orthologs or genes with the same function, ORTHOSCOPE uses the concept of an orthogroup. We define an orthogroup as follows: a set of genes descended from a single gene in the last common ancestor of all the species being considered. This definition is similar to that of Emms and Kelly (2015), but differs in that we use the species tree to identify the common ancestor.

Dependencies:
BLAST v2.7.1+
MAFFT v7.356b
trimAl 1.2rev59
PAL2NAL v13
ape in R, Version5.0
FastME v2.0 for amino acid analyses
Notung-2.9

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.

Queries.
Taxon sampling.
To count Fads2 gene copies, these sequences were used for "Comparing gene and species trees" mode of "Focal group Actinopterygii".

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 Queries For queries, CesA gene sequences were separated into 2 parts: CesA (before TM7) and GH6 (after TM7) domains. Taxon sampling In this paper, maximum likelihood trees were estimated according to the process described in "Tree Estimation of Orthogroup Members". See below.

Inoue and Satoh (2019)

Download query sequences from NCBI/Ensembl

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

The sequence used for rooting the gene tree is determined by a two-step rule:
(1) Species selection: Among the species included in the analysis, the sequence from the species ranked highest in the species list on the top page.
(2) Sequence selection: Among sequences from the same gene model, the sequence most distantly related to the query sequence (or the topmost query sequence when multiple queries are provided), as determined by the lowest BLAST score.

Analysis pipeline with example data: DeuterostomeBra_2ndAnalysis.zip

Installing dependencies

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

IQ-TREE

Available here: https://iqtree.github.io

Install the latest version according to the above site.

Add the directory path to your PATH, for example:

export PATH=$PATH:~/bin

Available here: https://mafft.cbrc.jp/alignment/software/ . After compilation, set your PATH following this site.

Available here: http://trimal.cgenomics.org/downloads

Move into trimAl/source, type make, and then copy the executable:

make
cp trimal ~/bin

Available here: http://www.bork.embl.de/pal2nal/#Download

Change the permission of the Perl script and copy it:

chmod 755 pal2nal.pl
cp pal2nal.pl ~/bin

R (3.5.2) is available from CRAN. By installing R, rscript is installed automatically.

Install APE in R by typing the following command in the R console:

install.packages("ape")

Tree estimation

Using the downloaded pipeline, the second gene trees are estimated as follows:

• Based on the estimated rearranged NJ tree, users should manually select coding sequences of the orthogroup and outgroups. The pipeline then starts subsequent analyses.
• Selected sequences are aligned using MAFFT (Katoh et al. 2005).
• Multiple sequence alignments are trimmed using trimAl (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).
• Phylogenetic analysis is performed using IQ-TREE 2 (Minh et al. 2020),

The actual process is as follows:

1. Decompress DeuterostomeBra_2ndAnalysis.zip, open the DeuterostomeBra_2ndAnalysis directory, and extract 100_2ndTree.tar.gz.
2. Select an appropriate outgroup and orthogroup members and save them as 010_candidates_nucl.txt. The outgroup sequence should be placed at the top of the alignment. Additional sequences can be included.

3. Change directory to 100_2ndTree.
4. Run the pipeline:

./100_estimate2ndTree.py

5. The ML tree is automatically saved.

Here, we demonstrate this procedure using vertebrate ALDH1A and actinopterygian PLCB1 (Inoue and Satoh, 2019).

1. Download Coregonus lavaretus TSA file (GFIG00000000.1) from NCBI.
2. Translate raw sequences into amino acid and coding sequences using TransDecoder.

   ./TransDecoder.LongOrfs -t GFIG01.1.fsa_nt

3. Create blast databases using BLAST+.

   makeblastdb -in longest_orfs.pep -dbtype prot -parse_seqids
   makeblastdb -in longest_orfs.cds -dbtype nucl -parse_seqids

4. Perform a BLASTP search against amino acid database.

   blastp -query query.txt -db longest_orfs.pep -num_alignments 10 -evalue 1e-12 -out 010_out.txt

5. Retrieve BLAST top hit sequences from coding sequence file using their sequence IDs.

   blastdbcmd -db longest_orfs.cds -dbtype nucl -entry_batch queryIDs.txt -out 020_out.txt

Inoue J, 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.