Co-evolution-TRNP1-and-GI

This repository contains data files and scripts to reproduce the analyses and results presented in our paper.

Protein

All relevant scripts listed below can be found within the protein/scripts folder.

Protein-coding sequence collection

1. collect_coding_seqs2/run_ccs2_function.sh - blast wrapper to extract orthologous protein-coding sequences from genomes. If you want to do the blast search using our scripts, you will need to download genomes listed in table protein/data/TRNP1_source_genomes.csv

2. processingL_final.R - process our own primate sequence assemblies from targeted re-sequencing

3. collect_coding_seqs2/Ferret_transcr_assembly_steps.sh and collect_coding_seqs2/Ferret_cutContig_makeFa.R - process the re-sequenced ferret sequence

4. collect_coding_seqs.R - gather the orthologous TRNP1 protein-coding sequences from all included sources. Intersect with the available trait data. Save sequences and traits for the downstream analyses

Evolutionary analysis of TRNP1 coding-sequence

Multiple Alignments with PRANK (v150803)

align_with_prank.sh - protein-coding sequence alignment

PAML (v4.8)

First, run PAML site models as described in the README from folder PAML. select_sign_sites_PAML_M8.R - pull out the identified sites under positive selection

COEVOL (v1.4)

1. run_coevol.sh - wrapper to run Coevol; finish_coevol.sh - wrapper to stop Coevol and generate summaries

2. summarize_coevol_output_TRNP1.R - access the estimated omega, correlations and posterior probabilities

Scripts for evolutionary analysis of control proteins can be found in a separate folder other_protein_alignments where there is a separate README on this part.

Analysis of NPC proliferation assay

proliferation_analysis.R - gather proliferation assay data, estimate proliferation rates using logistic regression, infer association with brain size and GI using PGLS

Regulation

All relevant scripts listed below can be found within the regulation/scripts folder.

MPRA assay

1. MPRA/MPRA_sequences.R - identify and collect orthologous TRNP1 CRE sequences across mammals from our sequenced data as well as published genomes

2. MPRA/MPRA_oligolib_construction.R - MPRA design - using a sliding window, construct enhancer tiles based on the orthologous CRE sequences from the previous script to test within the MPRA assay

3. MPRA/preprocessing MPRA count pre-processing. Extract reporter gene expression counts for each included enhancer tile. This folder contains a README with further details

4. MPRA/collect_MPRA_fastas.R - separate and save the relevant sequences from each of the 7 CRE regions, align using MAFFT (v7.407)

5. MPRA/MPRA_analysis.R - filter and summarize CRE activities. Plug into PGLS and compare to brain mass and gyrification

6. MPRA/combine_dnds_intron.R - combine TRNP1 protein evolution rates inferred using Coevol with the intron activity across catharrines within the same model

Transcription factor analysis

1. TFs/download_motifs_JASPAR2020.R - download PWMs and motif clustering from JASPAR 2020, transform PWMs for Cluster-Buster

2. TFs/MPRNAseq_NPC.yaml - zUMIs (v2.5.4) yaml file for mapping RNA-seq reads from NPCs. Input raw data for this processing can be accessed under E-MTAB-9951

3. TFs/TF_expression_analysis.R - find the expressed transcription factors in our NPCs (from bulk RNA-seq data). Run Cluster-Buster (Jun 13 2019) on the intron sequences including only the PWMs of the expressed TFs to identify overrepresented motifs

4. TFs/PGLS_motifs.R - investigate binding score assocation with intron CRE activity and GI among the 22 most abundant motifs on the intron sequence using PGLS

Tree construction: regulation/scripts/MPRA/tree_construction.R

Throughout the workflow, we are using job scheduling system slurm (v0.4.3).

Aditional Tables

Primer sequences for the resequencing of putative Trnp1 cis-regulatory elements as well as for the MPRA can be found in oligo_sequences/. For more information on the different tables please have a look at the README oligo_sequences/README