Schuster C, Gabel A, Drost H-G, Leyser O, Grosse I, Meyerowitz E. Rapid evolution of gene expression patterns in flowering plants. bioRxiv (2024).
For dNdS computations first users need to install the orthologr package.
As a next step, please also install BLAST+ into the /usr/local/bin directory on your computer.
Please be aware that the following dN/dS computations might run several hours with 12 - 20 cores or even up to 1 - 1.5 days with 4 - 8 cores.
The coding sequence files of Arabidopsis thaliana have been retrieved from ENSEMBL via the biomartr package. First, users need to install the biomartr package.
# compute dN/dS table of A. thaliana vs. all other species
orthologr::map_generator_dnds(
query_file = "../DevSeq_data/DevSeq_CDS/Query_files/Athaliana.fa",
subjects_folder = "../DevSeq_data/DevSeq_CDS/Athaliana_subject_files",
eval = "1E-5", # e value threshold for ortholog detection
ortho_detection = "RBH", # use conservative method: BLAST best reciprocal hit
aa_aln_type = "pairwise",
aa_aln_tool = "NW", # use Needleman-Wunsch Algorithm for global codon alignment
codon_aln_tool = "pal2nal",
dnds_est_method = "Comeron", # use robust dN/dS estimation (Comeron's method)
min_qry_coverage_hsp = 30, # min query coverage of hsp >= 30% of initial query locus
min_qry_perc_identity = 30, # min percent identity of hsp >= 30% to initial query locus
output_folder = "../DevSeq_data/DevSeq_dNdS_maps/OrthologR/DevSeq_Plants/Query_Athaliana/",
comp_cores = 4
)
# retrieve pairwise ortho tables and detect for each gene locus exactly one
# representative splice variant that maximizes the sequence homology
# between the two orthologous loci
devseq_ortho_tables <- orthologr::generate_ortholog_tables_all(
dNdS_folder = "../DevSeq_data/DevSeq_dNdS_maps/OrthologR/DevSeq_Plants/Query_Athaliana",
annotation_file_query = "../DevSeq_data/DevSeq_GTF/Query_files/Athaliana.gtf",
annotation_folder_subject = "../DevSeq_data/DevSeq_GTF/Athaliana_subject_files",
output_folder = "../DevSeq_data/DevSeq_orthologs/OrthologR/DevSeq_Plants/",
output_type = "gene_locus",
format = c("gtf", "gtf")
)
# add scope column
devseq_ortho_tables <- dplyr::mutate(devseq_ortho_tables, scope = 1 - (abs(q_len - alig_length) / q_len))p_all_ortho_thresholds_devseq <- orthologr::plot_diverse_homology_thresholds(devseq_ortho_tables, species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
))
cowplot::save_plot(
"p_all_ortho_thresholds_devseq.pdf",
p_all_ortho_thresholds_devseq,
base_height = 14,
base_width = 18
)p_core_sets_multi <- orthologr::plot_diverse_homology_thresholds_core_orthologs(devseq_ortho_tables, species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), type = "gene_locus")
cowplot::save_plot(
"p_core_sets_multi_devseq.pdf",
p_core_sets_multi,
base_height = 9,
base_width = 14
)
p_core_sets_multsplice_variant_devseq <- orthologr::plot_diverse_homology_thresholds_core_orthologs(devseq_ortho_tables, species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), type = "both")
cowplot::save_plot(
"p_core_sets_multsplice_variant_devseq.pdf",
p_core_sets_multsplice_variant_devseq,
base_height = 9,
base_width = 14
)# use homology thresholds:
devseq_ortho_tables_final <- dplyr::filter(devseq_ortho_tables, qcovhsp >= 70, perc_identity >= 30, scope >= 0.7)
readr::write_tsv(devseq_ortho_tables_final, "devseq_ortho_tables_all_splice_variants.tsv", col_names = TRUE)
# retrieve the core set of orthologs in tidy data format
devseq_core_orthologs <-
orthologr::retrieve_core_orthologs(
ortho_tables = devseq_ortho_tables_final,
species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
)
)
readr::write_tsv(devseq_core_orthologs, "devseq_core_orthologs_representative_splice_variant.tsv", col_names = TRUE)
# look at the results
devseq_core_orthologs
# test that core set only includes unique query_IDs and subject_IDs
dplyr::summarize(
dplyr::group_by(devseq_core_orthologs, subject_species),
n_unique_query_gene_locus_id = length(unique(query_gene_locus_id)),
n_non_unique_query_gene_locus_id = length((query_gene_locus_id)),
n_unique_query_id = length(unique(query_id)),
n_non_unique_query_id = length((query_id)),
n_unique_subject_gene_locus_id = length(unique(subject_gene_locus_id)),
n_non_unique_subject_gene_locus_id = length((subject_gene_locus_id)),
n_unique_subject_id = length(unique(subject_id)),
n_non_unique_subject_id = length((subject_id))
)p_plot_pairwise_orthologs <- orthologr::plot_pairwise_orthologs(ortho_tables = devseq_ortho_tables_final,
species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
),
n_core_orthologs = length(unique(devseq_core_orthologs$query_gene_locus_id)))
cowplot::save_plot(
"p_plot_pairwise_orthologs_devseq.pdf",
p_plot_pairwise_orthologs,
base_height = 8,
base_width = 12
)
p_dNdS_5_Athaliana <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(devseq_ortho_tables_final, !is.na(dNdS), dNdS <= 5),
type = "dNdS",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), xlab = "dNdS"
)
p_alig_length <- metablastr::gg_species_feature_blast_tbl(devseq_ortho_tables_final,
type = "alig_length",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), xlab = "Alignment length in amino acids (BLAST hits)"
)
p_perc_identity <- metablastr::gg_species_feature_blast_tbl(devseq_ortho_tables_final,
type = "perc_identity",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), xlab = "Percent Identity (BLAST hits)"
)
p_ortholog_divergence_devseq <- gridExtra::grid.arrange(p_alig_length, p_dNdS_5_Athaliana, p_perc_identity, ncol = 1)
cowplot::save_plot(
"p_ortholog_divergence_devseq.pdf",
p_ortholog_divergence_devseq,
base_height = 20,
base_width = 12
)
p_dNdS_5_devseq <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(devseq_ortho_tables_final, !is.na(dNdS), dNdS <= 5),
type = "dNdS",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), xlab = "dNdS"
)
p_dN_5_devseq<- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(devseq_ortho_tables_final, !is.na(dNdS), dNdS <= 5),
type = "dN",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), xlab = "dN"
)
p_dS_5_devseq <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(devseq_ortho_tables_final, !is.na(dNdS), dNdS <= 5),
type = "dS",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), xlab = "dS"
)
p_dN_plus_dS_5_devseq <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(devseq_ortho_tables_final, !is.na(dNdS), dNdS <= 5),
type = "dN+dS",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
), xlab = "dN+dS"
)
p_all_dNdS_devseq <- gridExtra::grid.arrange(p_dN_5_devseq, p_dS_5_devseq, p_dNdS_5_devseq, p_dN_plus_dS_5_devseq, nrow = 2)
cowplot::save_plot(
"p_all_dNdS_devseq.pdf",
p_all_dNdS_devseq,
base_height = 16,
base_width = 20
)# compute dN/dS table of A. thaliana vs. all other species
orthologr::map_generator_dnds(
query_file = "../DevSeq_data/EvoSeq_CDS/Query_files/Athaliana.fa",
subjects_folder = "../DevSeq_data/EvoSeq_CDS/Athaliana_subject_files",
eval = "1E-5", # e value threshold for ortholog detection
ortho_detection = "RBH", # use conservative method: BLAST best reciprocal hit
aa_aln_type = "pairwise",
aa_aln_tool = "NW", # use Needleman-Wunsch Algorithm for global codon alignment
codon_aln_tool = "pal2nal",
dnds_est_method = "Comeron", # use robust dN/dS estimation (Comeron's method)
min_qry_coverage_hsp = 70, # min query coverage of hsp >= 70% of initial query locus
min_qry_perc_identity = 30, # min percent identity of hsp >= 30% to initial query locus
output_folder = "../DevSeq_data/EvoSeq_dNdS_maps/OrthologR/EvoSeq_Plants/Query_Athaliana/",
comp_cores = 4
)
# retrieve pairwise ortho tables and detect for each gene locus exactly one
# representative splice variant that maximizes the sequence homology
# between the two orthologous loci
evoseq_ortho_tables <- orthologr::generate_ortholog_tables_all(
dNdS_folder = "../DevSeq_data/EvoSeq_dNdS_maps/OrthologR/EvoSeq_Plants/Query_Athaliana/",
annotation_file_query = "../DevSeq_data/EvoSeq_GTF/Query_files/Athaliana.gtf",
annotation_folder_subject = "../DevSeq_data/EvoSeq_GTF/Athaliana_subject_files",
output_folder = "../DevSeq_data/EvoSeq_orthologs/OrthologR/EvoSeq_Plants/",
output_type = "gene_locus",
format = c("gtf", "gtf")
)
readr::write_tsv(evoseq_ortho_tables, "evoseq_ortho_tables_all_splice_variants.tsv", col_names = TRUE)
# retrieve the core set of orthologs in tidy data format
evoseq_core_orthologs <-
orthologr::retrieve_core_orthologs(
ortho_tables = evoseq_ortho_tables,
species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
)
)
readr::write_tsv(evoseq_core_orthologs, "evoseq_core_orthologs_representative_splice_variant.tsv", col_names = TRUE)
# look at the results
evoseq_core_orthologs# compute dN/dS table of Human vs. all other species
orthologr::map_generator_dnds(
query_file = "../DevSeq_data/Brawand_CDS/Query_files/Human.fa",
subjects_folder = "../DevSeq_data/Brawand_CDS/Human_subject_files",
eval = "1E-5", # e value threshold for ortholog detection
ortho_detection = "RBH", # use conservative method: BLAST best reciprocal hit
aa_aln_type = "pairwise",
aa_aln_tool = "NW", # use Needleman-Wunsch Algorithm for global codon alignment
codon_aln_tool = "pal2nal",
dnds_est_method = "Comeron", # use robust dN/dS estimation (Comeron's method)
min_qry_coverage_hsp = 30, # min query coverage of hsp >= 30% of initial query locus
min_qry_perc_identity = 30, # min percent identity of hsp >= 30% to initial query locus
output_folder = "../DevSeq_data/Brawand_dNdS_maps/OrthologR/Brawand_Animals/Query_Human/",
comp_cores = 4
)
# retrieve pairwise ortho tables and detect for each gene locus exactly one
# representative splice variant that maximizes the sequence homology
# between the two orthologous loci
brawand_ortho_tables <- orthologr::generate_ortholog_tables_all(
dNdS_folder = "../DevSeq_data/Brawand_dNdS_maps/OrthologR/Brawand_Animals/Query_Human/",
annotation_file_query = "../DevSeq_data/Brawand_GTF/Query_files/Human.gtf",
annotation_folder_subject = "../DevSeq_data/Brawand_GTF/Human_subject_files",
output_folder = "../DevSeq_data/Brawand_orthologs/OrthologR/Brawand_Animals/",
output_type = "gene_locus",
format = c("gtf", "gtf")
)
brawand_ortho_tables <- dplyr::mutate(brawand_ortho_tables, scope = 1 - (abs(q_len - alig_length) / q_len))p_all_ortho_thresholds_brawand <- orthologr::plot_diverse_homology_thresholds(brawand_ortho_tables,
species_order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
))
cowplot::save_plot(
"p_all_ortho_thresholds_brawand.pdf",
p_all_ortho_thresholds_brawand,
base_height = 14,
base_width = 18
)p_core_sets_multi_brawand <- orthologr::plot_diverse_homology_thresholds_core_orthologs(brawand_ortho_tables, species_order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), type = "gene_locus")
cowplot::save_plot(
"p_core_sets_multi_brawand.pdf",
p_core_sets_multi_brawand,
base_height = 9,
base_width = 14
)
p_core_sets_multsplice_variant_brawand <- orthologr::plot_diverse_homology_thresholds_core_orthologs(brawand_ortho_tables, species_order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), type = "both")
cowplot::save_plot(
"p_core_sets_multsplice_variant_brawand.pdf",
p_core_sets_multsplice_variant_brawand,
base_height = 9,
base_width = 14
)# use homology thresholds:
brawand_ortho_tables_final <- dplyr::filter(brawand_ortho_tables, qcovhsp >= 70, perc_identity >= 30, scope >= 0.7)
readr::write_tsv(brawand_ortho_tables, "brawand_ortho_tables_all_splice_variants.tsv", col_names = TRUE)
# retrieve the core set of orthologs in tidy data format
brawand_core_orthologs <-
orthologr::retrieve_core_orthologs(
ortho_tables = brawand_ortho_tables_final,
species_order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
)
)
readr::write_tsv(brawand_core_orthologs, "brawand_core_orthologs_representative_splice_variant.tsv", col_names = TRUE)
# look at the results
brawand_core_orthologs
# test that core set only includes unique query_IDs and subject_IDs
dplyr::summarize(
dplyr::group_by(brawand_core_orthologs, subject_species),
n_unique_query_gene_locus_id = length(unique(query_gene_locus_id)),
n_non_unique_query_gene_locus_id = length((query_gene_locus_id)),
n_unique_query_id = length(unique(query_id)),
n_non_unique_query_id = length((query_id)),
n_unique_subject_gene_locus_id = length(unique(subject_gene_locus_id)),
n_non_unique_subject_gene_locus_id = length((subject_gene_locus_id)),
n_unique_subject_id = length(unique(subject_id)),
n_non_unique_subject_id = length((subject_id))
)p_n_orthos_human <- orthologr::plot_pairwise_orthologs(ortho_tables = brawand_ortho_tables_final,
species_order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
),
n_core_orthologs = length(unique(brawand_core_orthologs$query_gene_locus_id)))
cowplot::save_plot(
"p_n_orthos_human_brawand.pdf",
p_n_orthos_human,
base_height = 8,
base_width = 12
)
p_dNdS_human <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(brawand_ortho_tables, !is.na(dNdS)),
type = "dN/dS",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), xlab = "dN/dS"
)
p_dNdS_5_Human_brawand <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(brawand_ortho_tables, !is.na(dNdS), dNdS <= 5),
type = "dN/dS",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), xlab = "dN/dS"
)
p_all_human <- gridExtra::grid.arrange(p_n_orthos_human, p_dNdS_human, p_dNdS_5_Human_brawand)
p_dN_5_human <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(brawand_ortho_tables, !is.na(dNdS), dNdS <= 5),
type = "dN",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), xlab = "dN"
)
p_dS_5_human <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(brawand_ortho_tables, !is.na(dNdS), dNdS <= 5),
type = "dS",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), xlab = "dS"
)
p_dN_plus_dS_5_human <- metablastr::gg_species_dnds_blast_tbl(
dplyr::filter(brawand_ortho_tables, !is.na(dNdS), dNdS <= 5),
type = "dN+dS",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), xlab = "dN+dS"
)
p_all_dNdS_human <- gridExtra::grid.arrange(p_dN_5_human, p_dS_5_human, p_dNdS_5_human, p_dN_plus_dS_5_human, nrow = 2)
cowplot::save_plot(
"p_all_dNdS_human.pdf",
p_all_dNdS_human,
base_height = 16,
base_width = 20
)
p_alig_length_brawand <- metablastr::gg_species_feature_blast_tbl(brawand_ortho_tables,
type = "alig_length",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), xlab = "Alignment Length (BLAST hits)"
)
p_perc_identity_brawand <- metablastr::gg_species_feature_blast_tbl(brawand_ortho_tables,
type = "perc_identity",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
), xlab = "Percent Identity (BLAST hits)"
)
p_ortholog_divergence_brawand <- gridExtra::grid.arrange(p_alig_length_brawand, p_dNdS_5_Human_brawand, p_perc_identity_brawand)
cowplot::save_plot(
"p_ortholog_divergence_brawand.pdf",
p_ortholog_divergence_brawand,
base_height = 20,
base_width = 12
)# translate all CDS sequences into protein sequences and check
# for all CDS sequences whether or not they are divisible by 3
orthologr::translate_cds_to_protein_all(input_folder = "../DevSeq_data/DevSeq_CDS/Query_files",
output_folder = "../DevSeq_data/DevSeq_Proteins",
delete_corrupt_cds = FALSE)
# for each translated protein file retrieve the longest isoform per gene locus
orthologr::retrieve_longest_isoforms_all(
proteome_folder = "../DevSeq_data/DevSeq_Proteins",
annotation_folder = "../DevSeq_data/DevSeq_GTF/Query_files",
output_folder = "../DevSeq_data/DevSeq_Proteins_longest_isoforms",
annotation_format = "gtf")
# run orthofinder2 on protein sequences with longest splice variants
# system(paste0("orthofinder -f ", ws_wrap(proteome_folder)," -t ", cores," -a ", cores," -S diamond -n ", basename(proteome_folder)))
orthologr::orthofinder2(proteome_folder = "../DevSeq_data/DevSeq_Proteins_longest_isoforms", comp_cores = 4)
### Visualize Pairwise Orthogroups
path_to_devseq_pairwise_orthos <- "../DevSeq_data/DevSeq_orthologs/Orthofinder2/DevSeq_Plants/Results_DevSeq_Proteins_longest_isoforms/Orthologues/Orthologues_Athaliana/"
devseq_pairwise_files <- list.files(path_to_devseq_pairwise_orthos)
devseq_pairwise_df <- lapply(file.path(path_to_devseq_pairwise_orthos, devseq_pairwise_files), readr::read_tsv)
names(devseq_pairwise_df) <- c("Alyrata", "Bdistachyon", "Crubella", "Esalsugineum", "Mtruncatula", "Thassleriana")
devseq_pairwise_n_orthogroups <- lapply(devseq_pairwise_df, nrow)
devseq_pairwise_df <- tibble::tibble(subject_species = names(devseq_pairwise_n_orthogroups), n_orthologs = unlist(devseq_pairwise_n_orthogroups))
devseq_pairwise_df$subject_species <- factor(devseq_pairwise_df$subject_species,
levels = c("Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"))
### retrieve core orthogroups
og_file_devseq <- "../DevSeq_data/DevSeq_orthologs/Orthofinder2/DevSeq_Plants/Results_DevSeq_Proteins_longest_isoforms/Orthogroups/Orthogroups.tsv"
sc_file_devseq <- "../DevSeq_data/DevSeq_orthologs/Orthofinder2/DevSeq_Plants/Results_DevSeq_Proteins_longest_isoforms/Orthogroups/Orthogroups_SingleCopyOrthologues.txt"
DevSeq_OF2_CoreOrthologs <- orthologr::orthofinder2_retrieve_core_orthologs(orthogroups_file = og_file_devseq, single_copy_file = sc_file_devseq)
### visualize pairwise orthologs
devseq_plot <-
metablastr::gg_pairwise_orthologs_line(devseq_pairwise_df,
vline = nrow(DevSeq_OF2_CoreOrthologs),
ymax = 26000,
title = "Orthofinder2: Pairwise genome comparisons: A. thaliana vs. Subject Species")
# store DevSeq Orthofinder2 core orthogroups
readr::write_tsv(DevSeq_OF2_CoreOrthologs, "devseq_OF2_CoreOrthologs.tsv",col_names = TRUE)
cowplot::save_plot(
"p_orthofinder2_orthologs_devseq.pdf",
devseq_plot,
base_height = 8,
base_width = 12
)Extracting coding sequences from genome and gtf files.
# Bonobo
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Pan_paniscus.panpan1.1.92.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Pan_paniscus.panpan1.1.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Bonobo.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)
# Chicken
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Gallus_gallus.Gallus_gallus-5.0.92.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Gallus_gallus.Gallus_gallus-5.0.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Chicken.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)
# Chimp
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Pan_troglodytes.Pan_tro_3.0.92.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Pan_troglodytes.Pan_tro_3.0.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Chimp.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)
# Gorilla
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Gorilla_gorilla.gorGor4.92.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Gorilla_gorilla.gorGor4.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Gorilla.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)
# Human
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Homo_sapiens.GRCh38.92_ensembl.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Homo_sapiens.GRCh38.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Human.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)
# Macaque
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Macaca_mulatta.Mmul_8.0.1.92.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Macaca_mulatta.Mmul_8.0.1.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Macaque.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)
# Mouse
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Mus_musculus.GRCm38.92.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Mus_musculus.GRCm38.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Mouse.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)
# Orangutan
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Pongo_abelii.PPYG2.92.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Pongo_abelii.PPYG2.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Orangutan.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)
# Opossum
extractSeqsFromAnnotation(
annotation = "../DevSeq_data/Brawand_GTF/Monodelphis_domestica.monDom5.92.gtf",
genome_fasta = "../DevSeq_data/Brawand_Genomes/Monodelphis_domestica.monDom5.dna.toplevel.fa",
outputFile = "../DevSeq_data/Brawand_CDS/Opossum.fa",
format = "gtf",
metaFeature = "tx",
feature = "CDS"
)# translate all CDS sequences into protein sequences and check
# for all CDS sequences whether or not they are divisible by 3
orthologr::translate_cds_to_protein_all(input_folder = "../DevSeq_data/Brawand_CDS/Query_files",
output_folder = "../DevSeq_data/Brawand_Proteins",
delete_corrupt_cds = FALSE)
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Gallus_gallus.Gallus_gallus-5.0.92.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Chicken.gtf")
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Gorilla_gorilla.gorGor4.92.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Gorilla.gtf")
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Homo_sapiens.GRCh38.92_ensembl.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Human.gtf")
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Macaca_mulatta.Mmul_8.0.1.92.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Macaque.gtf")
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Monodelphis_domestica.monDom5.92.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Opossum.gtf")
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Mus_musculus.GRCm38.92.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Mouse.gtf")
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Pan_paniscus.panpan1.1.92.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Bonobo.gtf")
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Pan_troglodytes.Pan_tro_3.0.92.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Chimp.gtf")
file.rename(from = "../DevSeq_data/Brawand_GTF/Query_files/Pongo_abelii.PPYG2.92.gtf", to = "../DevSeq_data/Brawand_GTF/Query_files/Orangutan.gtf")
# for each translated protein file retrieve the longest isoform per gene locus
orthologr::retrieve_longest_isoforms_all(
proteome_folder = "../DevSeq_data/Brawand_Proteins",
annotation_folder = "../DevSeq_data/Brawand_GTF/Query_files",
output_folder = "../DevSeq_data/Brawand_Proteins_longest_isoforms",
annotation_format = "gtf")
# run orthofinder2 on protein sequences with longest splice variants
# system(paste0("orthofinder -f ", ws_wrap(proteome_folder)," -t ", cores," -a ", cores," -S diamond -n ", basename(proteome_folder)))
orthologr::orthofinder2(proteome_folder = "../DevSeq_data/Brawand_Proteins_longest_isoforms", comp_cores = 4)
# Brawand Core Orthologs
og_file_brawand <- "../DevSeq_data/Brawand_orthologs/Orthofinder2/Results_Brawand_Proteins_longest_isoforms/Orthogroups/Orthogroups.tsv"
sc_file_brawand <- "../DevSeq_data/Brawand_orthologs/Orthofinder2/Results_Brawand_Proteins_longest_isoforms/Orthogroups/Orthogroups_SingleCopyOrthologues.txt"
Brawand_OF2_CoreOrthologs <- orthologr::orthofinder2_retrieve_core_orthologs(orthogroups_file = og_file_brawand, single_copy_file = sc_file_brawand)
readr::write_tsv(Brawand_OF2_CoreOrthologs, "brawand_OF2_CoreOrthologs.tsv",col_names = TRUE)
### Brawand Pairwise Orthologs
Brawand_Statistics_PerSpecies <- readr::read_tsv("~/Desktop/Projects/orthofinder/orthofinder_results/Animals_Result/Results_Jan30/Comparative_Genomics_Statistics/Statistics_PerSpecies.tsv", n_max = 10)
names(Brawand_Statistics_PerSpecies)[1] <- "Type"
path_to_brawand_pairwise_orthos <- "../DevSeq_data/Brawand_orthologs/Orthofinder2/Results_Brawand_Proteins_longest_isoforms/Orthologues/Orthologues_Human"
brawand_pairwise_files <- list.files(path_to_brawand_pairwise_orthos)
brawand_pairwise_df <- lapply(file.path(path_to_brawand_pairwise_orthos, brawand_pairwise_files), readr::read_tsv)
names(brawand_pairwise_df) <- c("Bonobo", "Chicken", "Chimp", "Gorilla", "Macaque", "Mouse", "Opossum", "Orangutan")
brawand_pairwise_n_orthogroups <- lapply(brawand_pairwise_df, nrow)
brawand_pairwise_df <- tibble::tibble(subject_species = names(brawand_pairwise_n_orthogroups), n_orthologs = unlist(brawand_pairwise_n_orthogroups))
brawand_pairwise_df$subject_species <- factor(brawand_pairwise_df$subject_species,
levels = c("Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"))
brawand_plot <- metablastr::gg_pairwise_orthologs_line(brawand_pairwise_df, vline = nrow(Brawand_OF2_CoreOrthologs), title = "Orthofinder2: Pairwise genome comparisons: Human vs. Subject Species")
cowplot::save_plot(
"p_orthofinder2_orthologs_brawand.pdf",
brawand_plot,
base_height = 8,
base_width = 12
)We used *_No_TE_genes_tpm_sample_names.csv files.
### Use E-value threshold 1E-2
# gene loci lncRNAs
lnc_map_1e2 <- orthologr::map_generator_lnc(
query_file = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Query_files/Athaliana.fa",
subjects_folder = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Athaliana_subject_files/",
eval = "1E-2", # e value threshold for ortholog detection
ortho_detection = "RBH", # use conservative method: BLAST best reciprocal hit
output_folder = "../DevSeq_data/DevSeq_lncRNA_orthologs/lncRNA_gene_loci_1e2",
min_qry_coverage_hsp = 30,
min_qry_perc_identity = 30,
comp_cores = 4
)
devseq_core_orthologs_lnc_1e2 <-
orthologr::lnc_map_core_orthologs(
lnc_map = lnc_map_1e2,
species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
)
)
all_lncRNAs_df_1e2 <- orthologr::lnc_map_counts(lnc_map_1e2, species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
))
### Use E-value threshold 1E-3
# gene loci lncRNAs
lnc_map_1e3 <- orthologr::map_generator_lnc(
query_file = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Query_files/Athaliana.fa",
subjects_folder = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Athaliana_subject_files/",
eval = "1E-3", # e value threshold for ortholog detection
ortho_detection = "RBH", # use conservative method: BLAST best reciprocal hit
output_folder = "../DevSeq_data/DevSeq_lncRNA_orthologs/lncRNA_gene_loci_1e3",
min_qry_coverage_hsp = 30,
min_qry_perc_identity = 30,
comp_cores = 4
)
all_lncRNAs_df_1e3 <- orthologr::lnc_map_counts(lnc_map_1e3, species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
))
### Use E-value threshold 1E-5
# gene loci lncRNAs
lnc_map_1e5 <- orthologr::map_generator_lnc(
query_file = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Query_files/Athaliana.fa",
subjects_folder = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Athaliana_subject_files/",
eval = "1E-5", # e value threshold for ortholog detection
ortho_detection = "RBH", # use conservative method: BLAST best reciprocal hit
output_folder = "../DevSeq_data/DevSeq_lncRNA_orthologs/lncRNA_gene_loci_1e5",
min_qry_coverage_hsp = 30,
min_qry_perc_identity = 30,
comp_cores = 4
)
all_lncRNAs_df_1e5 <- orthologr::lnc_map_counts(lnc_map_1e5, species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
))
### Use E-value threshold 1E-7
# gene loci lncRNAs
lnc_map_1e7 <- orthologr::map_generator_lnc(
query_file = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Query_files/Athaliana.fa",
subjects_folder = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Athaliana_subject_files/",
eval = "1E-7", # e value threshold for ortholog detection
ortho_detection = "RBH", # use conservative method: BLAST best reciprocal hit
output_folder = "../DevSeq_data/DevSeq_lncRNA_orthologs/lncRNA_gene_loci_1e7",
min_qry_coverage_hsp = 30,
min_qry_perc_identity = 30,
comp_cores = 4
)
all_lncRNAs_df_1e7 <- orthologr::lnc_map_counts(lnc_map_1e7, species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
))
### Use E-value threshold 1E-10
# gene loci lncRNAs
lnc_map_1e10 <- orthologr::map_generator_lnc(
query_file = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Query_files/Athaliana.fa",
subjects_folder = "../DevSeq_data/DevSeq_lncRNAs/lncRNA_gene_loci/Athaliana_subject_files/",
eval = "1E-10", # e value threshold for ortholog detection
ortho_detection = "RBH", # use conservative method: BLAST best reciprocal hit
output_folder = "../DevSeq_data/DevSeq_lncRNA_orthologs/lncRNA_gene_loci_1e10",
min_qry_coverage_hsp = 30,
min_qry_perc_identity = 30,
comp_cores = 4
)
all_lncRNAs_df_1e10 <- orthologr::lnc_map_counts(lnc_map_1e10, species_order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
))
metablastr::gg_pairwise_orthologs_line(all_lncRNAs_df_1e2, title = "Number of orthologous lncRNAs: A. thaliana vs Subject Species") +
ggplot2::geom_line(ggplot2::aes(x = subject_species,
y = n_orthologs,
group = 1),
data = all_lncRNAs_df_1e3, size = 2) +
ggplot2::geom_point(size = 4, data = all_lncRNAs_df_1e3) +
ggplot2::geom_text(
ggplot2::aes(label = n_orthologs),
data = all_lncRNAs_df_1e3,
hjust = 0,
vjust = 2,
size = 2
) +
ggplot2::geom_line(ggplot2::aes(x = subject_species,
y = n_orthologs,
group = 1),
data = all_lncRNAs_df_1e5, size = 2) +
ggplot2::geom_point(size = 4, data = all_lncRNAs_df_1e5) +
ggplot2::geom_text(
ggplot2::aes(label = n_orthologs),
data = all_lncRNAs_df_1e5,
hjust = 0,
vjust = 2,
size = 2
) +
ggplot2::geom_line(ggplot2::aes(x = subject_species,
y = n_orthologs,
group = 1),
data = all_lncRNAs_df_1e7, size = 2) +
ggplot2::geom_point(size = 4, data = all_lncRNAs_df_1e7) +
ggplot2::geom_text(
ggplot2::aes(label = n_orthologs),
data = all_lncRNAs_df_1e7,
hjust = 0,
vjust = 2,
size = 2
) +
ggplot2::geom_line(ggplot2::aes(x = subject_species,
y = n_orthologs,
group = 1),
data = all_lncRNAs_df_1e10, size = 2) +
ggplot2::geom_point(size = 4, data = all_lncRNAs_df_1e10) +
ggplot2::geom_text(
ggplot2::aes(label = n_orthologs),
data = all_lncRNAs_df_1e10,
hjust = 0,
vjust = 2,
size = 2
)
p_all_lncRNA_plot <- metablastr::gg_pairwise_orthologs_line(all_lncRNAs_df_1e2, title = "Number of orthologous lncRNAs (only NATs): A. thaliana vs Subject Species")
cowplot::save_plot(
"p_all_lncRNA_plot.pdf",
p_all_lncRNA_plot,
base_height = 8,
base_width = 14
)anno_files <- file.path("/Volumes/BKP_1/BKP_1/devseq_data/DevSeq_GTF/Query_files", list.files("/Volumes/BKP_1/BKP_1/devseq_data/DevSeq_GTF/Query_files"))
genome_files <- file.path("/Volumes/BKP_1/BKP_1/devseq_data/DevSeq_Genomes/Query_files", list.files("/Volumes/BKP_1/BKP_1/devseq_data/DevSeq_Genomes/Query_files"))
# c(250, 500, 1000, 1500, 2000)
promotor_length_vector <- c(250, 500, 1000)
working_dir <- getwd()
dir.create("Promotor_Sequences")
message("Starting promotor extraction for promotor lengths: ", paste0(promotor_length_vector, collapse = ", "))
message(" and species: ", paste0(genome_files, collapse = ", "), " ...")
for (j in seq_len(length(promotor_length_vector))) {
message("Processing promotor length ", promotor_length_vector[j], " ...")
if (!file.exists(file.path("Promotor_Sequences", paste0("Promotor_length_", promotor_length_vector[j]))))
dir.create(file.path("Promotor_Sequences", paste0("Promotor_length_", promotor_length_vector[j])))
for (i in seq_len(length(anno_files))) {
species_name <-
unlist(stringr::str_split(basename(genome_files[i]), "[.]"))[1]
message("Processing species ", species_name, " ...")
setwd(file.path("Promotor_Sequences", paste0("Promotor_length_", promotor_length_vector[j])))
metablastr::extract_promotor_seqs_from_genome(
annotation_file = anno_files[i],
genome_file = genome_files[i],
annotation_format = "gtf",
promotor_length = promotor_length_vector[j]
)
setwd(working_dir)
cat("\n")
cat("\n")
}
message("Promotor extraction terminated successfully!")
}# compute pairwise Kimura DNA Distances for promotor sequences (250bp)
# of orthologous genes between A thaliana and all subject species
promotor_homology_ath_250 <-
orthologr::promotor_divergence_of_orthologous_genes(
promotor_folder = "Promotor_Sequences/Promotor_length_250/",
ortholog_tables_folder = "orthologs_by_gene_locus_qry_athaliana",
ortholog_promotor_seq_output = "ortholog_genes_promotor_seqs_250")
readr::write_delim(promotor_homology_ath_250, "ortholog_table_promotor_homology_ath_250.csv", delim = ";")
ortholog_table_promotor_homology_ath_250 <- readr::read_delim("/Volumes/BKP_1/BKP_1/devseq_data/DevSeq_Promotor_Analysis/ortholog_table_promotor_homology_ath_250.csv", delim = ";")
ortholog_table_promotor_homology_ath_250 <- dplyr::filter(ortholog_table_promotor_homology_ath_250, promotor_dist <= 10)
# compute pairwise Kimura DNA Distances for promotor sequences (500bp)
# of orthologous genes between A thaliana and all subject species
promotor_homology_ath_500 <-
orthologr::promotor_divergence_of_orthologous_genes(
promotor_folder = "Promotor_Sequences/Promotor_length_500/",
ortholog_tables_folder = "orthologs_by_gene_locus_qry_athaliana",
ortholog_promotor_seq_output = "ortholog_genes_promotor_seqs_500")
readr::write_delim(promotor_homology_ath_500, "ortholog_table_promotor_homology_ath_500.csv", delim = ";")
ortholog_table_promotor_homology_ath_500 <- readr::read_delim("/Volumes/BKP_1/BKP_1/devseq_data/DevSeq_Promotor_Analysis/ortholog_table_promotor_homology_ath_500.csv", delim = ";")
ortholog_table_promotor_homology_ath_500 <- dplyr::filter(ortholog_table_promotor_homology_ath_500, promotor_dist <= 10)
# compute pairwise Kimura DNA Distances for promotor sequences (1000bp)
# of orthologous genes between A thaliana and all subject species
promotor_homology_ath_1000 <-
orthologr::promotor_divergence_of_orthologous_genes(
promotor_folder = "Promotor_Sequences/Promotor_length_1000/",
ortholog_tables_folder = "orthologs_by_gene_locus_qry_athaliana",
ortholog_promotor_seq_output = "ortholog_genes_promotor_seqs_1000")
readr::write_delim(promotor_homology_ath_1000, "ortholog_table_promotor_homology_ath_1000.csv", delim = ";")
ortholog_table_promotor_homology_ath_1000 <- readr::read_delim("/Volumes/BKP_1/BKP_1/devseq_data/DevSeq_Promotor_Analysis/ortholog_table_promotor_homology_ath_1000.csv", delim = ";")
ortholog_table_promotor_homology_ath_1000 <- dplyr::filter(ortholog_table_promotor_homology_ath_1000, promotor_dist <= 10)### Promotor length 250
p_promotor_dist_250 <- metablastr::gg_species_promotor_dist_blast_tbl(
ortholog_table_promotor_homology_ath_250,
type = "promotor_dist",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
),
title = "Distributions of Pairwise Promotor Sequence Distance (250bp) Between A thaliana vs. Subjects",
xlab = "Kimura DNA Distance Between Promotors of Orthologous A thaliana Genes vs Subject Genes"
)
cowplot::save_plot(
"p_promotor_dist_250bp.pdf",
p_promotor_dist_250,
base_height = 8,
base_width = 14
)
### Promotor length 500
p_promotor_dist_500 <-metablastr::gg_species_promotor_dist_blast_tbl(
ortholog_table_promotor_homology_ath_500,
type = "promotor_dist",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
),
title = "Distributions of Pairwise Promotor Sequence Distance (500bp) Between A thaliana vs. Subjects",
xlab = "Kimura DNA Distance Between Promotors of Orthologous A thaliana Genes vs Subject Genes"
)
cowplot::save_plot(
"p_promotor_dist_500bp.pdf",
p_promotor_dist_500,
base_height = 8,
base_width = 14
)
### Promotor length 1000
p_promotor_dist_1000 <- metablastr::gg_species_promotor_dist_blast_tbl(
ortholog_table_promotor_homology_ath_1000,
type = "promotor_dist",
order = c(
"Alyrata",
"Crubella",
"Esalsugineum",
"Thassleriana",
"Mtruncatula",
"Bdistachyon"
),
title = "Distributions of Pairwise Promotor Sequence Distance (1000bp) Between A thaliana vs. Subjects",
xlab = "Kimura DNA Distance Between Promotors of Orthologous A thaliana Genes vs Subject Genes"
)
cowplot::save_plot(
"p_promotor_dist_1000bp.pdf",
p_promotor_dist_1000,
base_height = 8,
base_width = 14
)### Extract genes with promotor distance < 0.2 for PANTHER analysis (250bp)
readr::write_lines(unique(
dplyr::filter(
ortholog_table_promotor_homology_ath_250,
promotor_dist <= 0.2
)$query_gene_locus_id
), "gene_list_ortholog_table_promotor_homology_ath_250_smaller_0_2.txt")
### Extract genes with promotor distance < 0.2 for PANTHER analysis (500bp)
readr::write_lines(unique(
dplyr::filter(
ortholog_table_promotor_homology_ath_500,
promotor_dist <= 0.2
)$query_gene_locus_id
), "gene_list_ortholog_table_promotor_homology_ath_500_smaller_0_2.txt")
### Extract genes with promotor distance < 0.2 for PANTHER analysis (1000bp)
readr::write_lines(unique(
dplyr::filter(
ortholog_table_promotor_homology_ath_1000,
promotor_dist <= 0.2
)$query_gene_locus_id
), "gene_list_ortholog_table_promotor_homology_ath_1000_smaller_0_2.txt")Please make sure that you have enough memory (RAM) resources for running the following steps (> 100GB RAM) since the human genome file is >50GB.
anno_files <- file.path("/Volumes/BKP_1/BKP_1/devseq_data/Brawand_GTF/Query_files", list.files("/Volumes/BKP_1/BKP_1/devseq_data/Brawand_GTF/Query_files"))
genome_files <- file.path("/Volumes/BKP_1/BKP_1/devseq_data/Brawand_Genomes", list.files("/Volumes/BKP_1/BKP_1/devseq_data/Brawand_Genomes"))
# c(250, 500, 1000, 1500, 2000)
promotor_length_vector <- c(250, 500, 1000)
working_dir <- getwd()
dir.create("Promotor_Sequences_Brawand")
message("Starting promotor extraction for promotor lengths: ", paste0(promotor_length_vector, collapse = ", "))
message(" and species: ", paste0(genome_files, collapse = ", "), " ...")
for (j in seq_len(length(promotor_length_vector))) {
message("Processing promotor length ", promotor_length_vector[j], " ...")
if (!file.exists(file.path("Promotor_Sequences_Brawand", paste0("Promotor_length_", promotor_length_vector[j]))))
dir.create(file.path("Promotor_Sequences_Brawand", paste0("Promotor_length_", promotor_length_vector[j])))
for (i in seq_len(length(anno_files))) {
species_name <-
unlist(stringr::str_split(basename(genome_files[i]), "[.]"))[1]
message("Processing species ", species_name, " ...")
setwd(file.path("Promotor_Sequences_Brawand", paste0("Promotor_length_", promotor_length_vector[j])))
metablastr::extract_promotor_seqs_from_genome(
annotation_file = anno_files[i],
genome_file = genome_files[i],
annotation_format = "gtf",
promotor_length = promotor_length_vector[j]
)
setwd(working_dir)
cat("\n")
cat("\n")
}
message("Promotor extraction terminated successfully!")
}# compute pairwise Kimura DNA Distances for promotor sequences (250bp)
# of orthologous genes between Human and all subject species
promotor_homology_human_250 <-
orthologr::promotor_divergence_of_orthologous_genes(
promotor_folder = "Brawand_Promotor_Sequences/Promotor_length_250/",
ortholog_tables_folder = "orthologs_by_gene_locus_qry_human",
ortholog_promotor_seq_output = "Brawand_Promotor_Analysis/brawand_ortholog_genes_promotor_seqs_250")
readr::write_delim(promotor_homology_human_250, "Brawand_Promotor_Analysis/brawand_ortholog_table_promotor_homology_human_250.csv", delim = ";")
ortholog_table_promotor_homology_human_250 <- readr::read_delim("/Volumes/BKP_1/BKP_1/devseq_data/Brawand_Promotor_Analysis/brawand_ortholog_table_promotor_homology_human_250.csv", delim = ";")
ortholog_table_promotor_homology_human_250 <- dplyr::filter(ortholog_table_promotor_homology_human_250, promotor_dist <= 10)
# compute pairwise Kimura DNA Distances for promotor sequences (500bp)
# of orthologous genes between Human and all subject species
promotor_homology_human_500 <-
orthologr::promotor_divergence_of_orthologous_genes(
promotor_folder = "Brawand_Promotor_Sequences/Promotor_length_500/",
ortholog_tables_folder = "orthologs_by_gene_locus_qry_human",
ortholog_promotor_seq_output = "Brawand_Promotor_Analysis/brawand_ortholog_genes_promotor_seqs_500")
readr::write_delim(promotor_homology_human_500, "brawand_ortholog_table_promotor_homology_human_500.csv", delim = ";")
ortholog_table_promotor_homology_human_500 <- readr::read_delim("/Volumes/BKP_1/BKP_1/devseq_data/Brawand_Promotor_Analysis/brawand_ortholog_table_promotor_homology_human_500.csv", delim = ";")
ortholog_table_promotor_homology_human_500 <- dplyr::filter(ortholog_table_promotor_homology_human_500, promotor_dist <= 10)
# compute pairwise Kimura DNA Distances for promotor sequences (1000bp)
# of orthologous genes between Human and all subject species
promotor_homology_human_1000 <-
orthologr::promotor_divergence_of_orthologous_genes(
promotor_folder = "Brawand_Promotor_Sequences/Promotor_length_1000/",
ortholog_tables_folder = "orthologs_by_gene_locus_qry_human",
ortholog_promotor_seq_output = "Brawand_Promotor_Analysis/brawand_ortholog_genes_promotor_seqs_1000")
readr::write_delim(promotor_homology_human_1000, "brawand_ortholog_table_promotor_homology_human_1000.csv", delim = ";")
ortholog_table_promotor_homology_human_1000 <- readr::read_delim("/Volumes/BKP_1/BKP_1/devseq_data/Brawand_Promotor_Analysis/brawand_ortholog_table_promotor_homology_human_1000.csv", delim = ";")
ortholog_table_promotor_homology_human_1000 <- dplyr::filter(ortholog_table_promotor_homology_human_1000, promotor_dist <= 10)### Promotor length 250
p_human_promotor_dist_250 <- metablastr::gg_species_promotor_dist_blast_tbl(
ortholog_table_promotor_homology_human_250,
type = "promotor_dist",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
),
title = "Distributions of Pairwise Promotor Sequence Distance (250bp) Between Human vs. Subjects",
xlab = "Kimura DNA Distance Between Promotors of Orthologous Human Genes vs Subject Genes"
)
cowplot::save_plot(
"p_brawand_promotor_dist_250.pdf",
p_human_promotor_dist_250,
base_height = 8,
base_width = 14
)
### Promotor length 500
p_human_promotor_dist_500 <-metablastr::gg_species_promotor_dist_blast_tbl(
ortholog_table_promotor_homology_human_500,
type = "promotor_dist",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
),
title = "Distributions of Pairwise Promotor Sequence Distance (500bp) Between Human vs. Subjects",
xlab = "Kimura DNA Distance Between Promotors of Orthologous Human Genes vs Subject Genes"
)
cowplot::save_plot(
"p_brawand_promotor_dist_500bp.pdf",
p_human_promotor_dist_500,
base_height = 8,
base_width = 14
)
### Promotor length 1000
p_human_promotor_dist_1000 <- metablastr::gg_species_promotor_dist_blast_tbl(
ortholog_table_promotor_homology_human_1000,
type = "promotor_dist",
order = c(
"Chimp",
"Bonobo",
"Gorilla",
"Orangutan",
"Macaque",
"Mouse",
"Opossum",
"Chicken"
),
title = "Distributions of Pairwise Promotor Sequence Distance (1000bp) Between Human vs. Subjects",
xlab = "Kimura DNA Distance Between Promotors of Orthologous Human Genes vs Subject Genes"
)
cowplot::save_plot(
"p_brawand_promotor_dist_1000bp.pdf",
p_human_promotor_dist_1000,
base_height = 8,
base_width = 14
)The DevSeqR package allows users to reproduce all analyses and to perform additional exploratory data analysis using the DevSeq dataset.
# install.packages("devtools")
# install the current version of DevSeqR on your system
library(devtools)
install_github("HajkD/DevSeqR", build_vignettes = TRUE, dependencies = TRUE)# load DevSeqR package
library(DevSeqR)
# import experimental design information for the DevSeq dataset
DevSeqSample <- load.sample.info()
DevSeqSample Sample_ID DevSeq_ID Species Tissue Description
<dbl> <chr> <chr> <chr> <chr>
1 1 10-12 A.thaliana root root, root tip (top 0.5-1mm)
2 2 13-15 A.thaliana root root, maturation zone (2-3mm piece above root tip)
3 3 106-108 A.thaliana root root, whole root (3mm piece including root tip)
4 4 1-3 A.thaliana root root, whole root
5 5 7-9 A.thaliana root root, whole root
6 6 4-6 A.thaliana root root, whole root
7 7 16-18 A.thaliana stem hypocotyl
8 8 22-24 A.thaliana stem 3rd internode (0.5cm piece)
9 9 19-21 A.thaliana stem 2nd internode (1cm piece)
10 10 25-27 A.thaliana stem 1st internode (1cm piece)
... with 130 more rows, and 5 more variables: Age <chr>, Substrate <chr>,
Photoperiod <chr>, Replicates <dbl>, Sample_size <chr>