Selection in coral mitogenomes, with insights into adaptations in the deep sea

This repository contains supporting data and code for assessing positive selection in deep- and shallow- water species of corals. The data and workflow provided within this repository are consistent with:

Ramos, N. I., DeLeo, D. M., Horowitz, J., McFadden, C. S. Quattrini, A. M. Selection in coral mitogenomes, with insights into adaptations in the deep sea. Scientific Reports (In Review).

A portion of analyses were conducted on the Smithsonian High Performance Computing Cluster. Smithsonian Institution. https://doi.org/10.25572/SIHPC

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The following programs were used to complete this analysis

• Iqtree to create Maximum-likelihood tree
• Mitos2 to annotate sequences
• MAFFT to align sequences
• Phyluce to concatenate protein-coding genes
• Codeml for positive selection analysis

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Data Collection and Preparation

1. Download coral mitogenome data from GenBank

List of accession IDs used in this study can be found in Supplementary Info Table S1.

2. Annotate mitogenomes to identify protein-coding genes

Mitogenomes annotated using Mitos2 to identify protein coding genes, tRNAs, and rRNAs

• reference NCBI RefSeq 89 Metazoa
• genetic code 5 (invertebrate)

3. Align protein-coding genes separately with MAFFT

• L-INS-I method

4. Phylogenetic Trees

• Create a species tree for all of the sequences in alignment using IQtree

iqtree2 -s filename.phy -m GTR+G -bb 1000 to run with the GTR+G model

iqtree2 -s filename.phy -m TESTNEW -bb 1000 to run with ModelFinder and construct trees with the best-fit model

• can add rcluster10 for relaxed clustering to speed up computation (means only top 10% partitiion schemes are considered to save computations)

For more information on how to run IQtree: Begginer's tuturial

IQtree will produce three outfiles: filename.phy.treefile is the maximum-likelihood tree in NEWICK format which can be visualized by Figtree, and what you will need to run codeml

Unroot tree on R

• use Script_to_unroot_tree.R found in Code/

Remove extra support values from unrooted tree on R

• use Script_clean_unrooted_tree.R found in Code/

Labeling unrooted species tree

• Some models for codeml require specied branches on the species tree to be labeled for analysis. This allows separate ω's to be generated through different parameters of interest being assigned among the branches.
• For this project, both Branch models and Branch-site models required the species tree to be labeled. We defined the foreground branch as deep-water species and background branch as shallow-water species.
• To label unrooted species tree in newick format:
  • label foreground branches with a #1 inserted after the name/ID but before the semicolon and support value
  • example: DNA28_Letepsammia_superstes#1:0.0002563394,DNA41_Rhombopsammia_niphada#1:0.0003394101

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Running codeml

The following workflow was supported by modifications made to code presented in https://github.com/Santagata/Select_Test

PAML Manual

Files needed to run Codeml

• Control File
• Unrooted species tree in newick format
• Sequence file in PHYLIP format

To create control file using scripts

1. Download perl scripts from "Data/Create Control Files/"

2. Create a list of the alignment files that you wish to run for these models.

   • if all of the files end in the same extentions `ls *.phy > phylist.txt
   • make sure that the names/ids in file match the names on the tree

3. Create a list of the corresponding species tree file name

   • this list will repeat the treefile name as many times as there are alignments that are going to be tested
   • (i.e.) if you have 15 alignments to run and create control files for, list the species tree filename 15 times
   • seq 15 | awk '{print "SpeciesTree_Unrooted.tree"}' > treelist.txt

4. Run perl scripts below to create the control files for each model

   • CODEML_TREE_ALN_ALT.pl:creates control file for alternative model (Model A)

   • CODEML_TREE_ALN_NULL.pl:creates control file for null model

   • Note: These perl scripts specify for the invertebrate genetic code icode = 4

5. Edit scripts to create control files for other models by modifying the line in CODEML_TREE_ALN_ALT.pl that you are changing the paramenter for.

   "one-ratio" (M0)

   • model = 0; modify the line system("echo model = 2 >> $a_files[$i].ctl"); to system("echo model = 0 >> $a_files[$i].ctl");
   • NSsites = 0; modify the line system("echo NSsites = 2 >> $a_files[$i].ctl"); to system("echo NSsites = 0 >> $a_files[$i].ctl");

   "two-ratios" (M2)

   • model = 2; keep line the same
   • NSsites = 0; modify the line system("echo NSsites = 2 >> $a_files[$i].ctl"); to system("echo NSsites = 0 >> $a_files[$i].ctl");

Run CODEML using perl script

1. Download perl script CODEML_CTL_RUN.pl

2. Change the line, system("/YOUR_PATH/codeml/paml4.9d/bin/codeml /YOUR_PATH/MCL_CTL_Files/$ctl_files[$i]\n"); to include the path to Codeml on your computer.

3. Make sure species tree is labeled and names/IDs match with those in the phylip files

4. Make a list of the control files that you are planning to run cat *.ctl > MCL_ALT_list.ctl cat *.ctl > MCL_NULL_list.ctl

5. Run as perl CODEML_CTL_RUN.pl MCL_ALT_list.ctl