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input_data/*sample_sheet.csv | ||
input_data/salmon_quantification_v38_gibbs/* | ||
external_tables/GSE125837_monolayer_rawcounts.txt.gz | ||
dna_methylation_arrays/* | ||
outputs/* |
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## --------------------------- | ||
## | ||
## Script name: | ||
## | ||
## Purpose of script: | ||
## | ||
## Author: Prakhar Bansal | ||
## | ||
## Date Created: 2021-09-08 | ||
## | ||
## Copyright (c) Prakhar Bansal, 2021 | ||
## Email: pbansal@uchc.edu | ||
## | ||
## --------------------------- | ||
## | ||
## Notes: | ||
## | ||
## | ||
## --------------------------- | ||
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## set working directory | ||
setwd(dirname(rstudioapi::getActiveDocumentContext()$path)) | ||
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## --------------------------- | ||
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## load up the packages we will need: (uncomment as required) | ||
require(tidyverse); theme_set(theme_classic() + theme(plot.title=element_text(hjust=0.5))) | ||
require(ggsci) | ||
library(zeallot) | ||
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## --------------------------- | ||
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output_dir <- "outputs/2024-02-17 21-39-53.641348 timepoint+dox_status+effective_genotype" | ||
sample_sheet_df <- read_csv(paste0(output_dir, "/tables/sample_sheet_df.csv.gz")) |> | ||
mutate(cell_type = "ipscs") | ||
neural_output_dir <- paste0("../salmon_pipeline_with_stefan/", | ||
(read_csv(paste0(output_dir, "/params.csv.gz")) |> | ||
filter(parameter == "neural_output_dir") |> | ||
pull(value))[1]) | ||
sample_sheet_df_neural <- read_csv(paste0(neural_output_dir, "/tables/sample_sheet_df.csv.gz")) |> | ||
mutate( | ||
cell_line = str_replace_all(cell_line, "C(?=(1|4|7))", "rtTA_XIST_c"), | ||
`Cell Line` = str_replace_all(cell_line, "_c", " c") |> | ||
str_replace_all("_", "-"), | ||
dox_status = case_when(dox_status == "wd" ~ "withdrawal", | ||
T ~ dox_status), | ||
has_transgene = case_when(cell_line %in% c("198_1", "198_5") ~ F, | ||
T ~ T) | ||
)|> | ||
mutate(cell_type = "neurons") | ||
gene_info_df <- read_csv("tables/gene_info_df.csv.gz") | ||
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comb_sample_sheet_df <- bind_rows(sample_sheet_df, sample_sheet_df_neural) | ||
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chr21_allelic_counts_df <- read_tsv("phaser_output_all_chr21/combined_bams_gene_ae.txt", | ||
col_types = cols(contig=col_character(), | ||
variants=col_character())) |> | ||
filter(contig == "21" & | ||
totalCount > 0) |> | ||
mutate(sample_id=str_extract(bam, "d[0-9]*")) |> | ||
inner_join(sample_sheet_df) | ||
write_csv(chr21_allelic_counts_df, "tables/chr21_allelic_counts_df.csv.gz") | ||
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chr21_allelic_counts_jan2024_df <- read_tsv("phaser_output_Jan2024/combined_bams_gene_ae.txt", | ||
col_types = cols(contig=col_character(), | ||
variants=col_character())) |> | ||
filter(contig == "21" & | ||
totalCount > 0) |> | ||
mutate(sample_id=str_extract(bam, "d[0-9]*")) |> | ||
right_join(comb_sample_sheet_df) | ||
write_csv(chr21_allelic_counts_df, "tables/chr21_allelic_counts_df_jan2024.csv.gz") | ||
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make_allelic_plot <- function(chr21_allelic_counts_df, allele_designation_df_byt21=F, keep_cell_type = "ipscs") { | ||
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chr21_allelic_counts_df <- chr21_allelic_counts_df |> | ||
filter(cell_type == keep_cell_type) | ||
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print(chr21_allelic_counts_df$sample_id |> unique()) | ||
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if(length(allele_designation_df_byt21) == 1 && allele_designation_df_byt21 == FALSE) { | ||
allele_designation_df_byt21 <- chr21_allelic_counts_df |> | ||
filter(`Cell Line` %in% c("198-1", "198-2", "198-5"), | ||
totalCount > 6) |> | ||
pivot_longer(cols=c(aCount, bCount), names_to = "allele", values_to = "count") |> | ||
group_by(name, allele, genotype) |> | ||
summarise(mean_ratio=mean(count/totalCount)) |> | ||
ungroup() |> | ||
tidyr::pivot_wider(names_from = c("genotype", "allele"), values_from = "mean_ratio") |> | ||
na.omit() |> | ||
mutate(a_allele_diff=T21_aCount-D21_aCount, | ||
b_allele_diff=T21_bCount-D21_bCount, | ||
paternal_allele=case_when(abs(a_allele_diff) < .1 & abs(b_allele_diff) < .1 ~ "nodiff", | ||
a_allele_diff>0 & b_allele_diff < 0 ~ "bCount", | ||
a_allele_diff<0 & b_allele_diff > 0 ~ "aCount", | ||
T ~ "undetermined"), | ||
maternal_allele=case_when(paternal_allele == "aCount" ~ "bCount", | ||
paternal_allele == "bCount" ~ "aCount")) |> | ||
filter(paternal_allele %in% c("aCount", "bCount")) |> | ||
pivot_longer(cols = c(paternal_allele, maternal_allele), names_to = "allele_type", values_to = "allele") | ||
} | ||
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print("ENSG00000154645.14" %in% allele_designation_df_byt21$name) | ||
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rtta_xist_ae_plot_df <- chr21_allelic_counts_df |> | ||
filter(!is.na(`Cell Line`) & totalCount >= 10) |> | ||
mutate(dox_status=str_replace(dox_status, "withdrawal", "w/d"), | ||
sample_type=case_when(has_transgene == T & dox_status == "no" ~ "T21XIST\n-dox", | ||
has_transgene == T & dox_status == "yes" ~ "T21XIST\n+dox", | ||
has_transgene == T & dox_status == "w/d" & timepoint == "6wk" ~ "T21XIST\n3w/d", | ||
has_transgene == T & dox_status == "w/d" & timepoint == "17wk" ~ "T21XIST\n3w/d", | ||
has_transgene == T & dox_status == "w/d" & timepoint == "9wk" ~ "T21XIST\n6w/d", | ||
has_transgene == T & dox_status == "w/d" & timepoint == "20wk" ~ "T21XIST\n6w/d", | ||
has_transgene == T & dox_status == "w/d" & is.na(timepoint) ~ "T21XIST\nw/d", | ||
cell_line == "198_5" ~ "T21", | ||
cell_line %in% c("198_1", "198_2") ~ "D21", | ||
T ~ cell_line)) |> | ||
pivot_longer(cols=c(aCount, bCount), names_to = "allele", values_to = "count") |> | ||
mutate(ratio=count/totalCount) |> | ||
inner_join(allele_designation_df_byt21, by=c("name", "allele")) |> | ||
group_by(sample_type, name, start, allele_type) |> | ||
summarise(mean_count=mean(count), | ||
mean_ratio=mean(ratio), | ||
totalCount=dplyr::first(totalCount)) |> | ||
ungroup() | ||
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ensembl_ids_to_keep <- rtta_xist_ae_plot_df |> | ||
left_join(gene_info_df, by=c("name"="ensembl_gene_id_version")) |> | ||
filter(allele_type=="paternal_allele" & | ||
gene_biotype %in% c("protein_coding")) |> | ||
pivot_wider(id_cols=c(name), names_from=sample_type, values_from=mean_ratio) |> | ||
na.exclude() |> | ||
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# dplyr::filter(`pb_no` < .9 & `pb_no` > .1) |> | ||
pull(name) | ||
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genes_sorted_by_position <- rtta_xist_ae_plot_df |> | ||
filter(name %in% ensembl_ids_to_keep) |> | ||
arrange(start) |> | ||
pull(name) |> | ||
unique() | ||
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#potential escapees | ||
potential_escapees <- rtta_xist_ae_plot_df |> | ||
filter(sample_type == "T21XIST\n+dox" & | ||
name %in% ensembl_ids_to_keep & | ||
allele_type == "paternal_allele" & | ||
mean_ratio >= 1/6) |> | ||
left_join(gene_info_df, by=c("name"="ensembl_gene_id_version")) |> | ||
pull(name) | ||
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plt <- ggplot(rtta_xist_ae_plot_df |> | ||
filter(name %in% ensembl_ids_to_keep) |> | ||
mutate(sample_type1 = case_when(str_detect(sample_type, "T21XIST") ~ "T21XIST", | ||
T ~ sample_type), | ||
sample_type2 = case_when(!str_detect(sample_type, "T21XIST") ~ "", | ||
T ~ str_extract(sample_type, "\\-dox|\\+dox|3w\\/d|6w\\/d|w\\/d"))) |> | ||
left_join(gene_info_df, by=c("name"="ensembl_gene_id_version")), | ||
aes(x=name, y=mean_ratio, fill=allele_type, totalCount=totalCount, gene_name=hgnc_symbol)) + | ||
geom_col(position=position_fill(), width=.8) + | ||
scale_x_discrete(limits=genes_sorted_by_position) + | ||
scale_fill_d3(limits=c("maternal_allele", "paternal_allele"), labels=c("M", "P")) + | ||
ggh4x::facet_nested(rows = vars(factor(sample_type1, levels = c("D21", "T21", "T21XIST")), | ||
factor(sample_type2, levels = c("","-dox", "+dox", "3w/d", "6w/d", "w/d"))), | ||
strip = ggh4x::strip_nested(clip = "off", size = "variable"), | ||
nest_line = element_line(linetype = 1)) + | ||
#strip=ggh4x::strip_nested(clip="off") | ||
# facet_grid(rows = ) + | ||
scale_y_continuous(breaks = c(0,.5,1)) + | ||
geom_hline(yintercept=.5, size=.5) + | ||
geom_hline(yintercept=1/3, linetype="dashed", size=.3) + | ||
labs(title="HSA21 Protein Coding Gene Allelic Expression", | ||
x="Gene", | ||
y="Fraction of Expression", | ||
fill="Allele") + | ||
theme(axis.ticks.x = element_blank(), | ||
axis.text.x = element_blank(), | ||
legend.position = "top", | ||
legend.title = element_text(margin = margin(r=3, unit="mm")), | ||
legend.text = element_text(margin = margin(r=2, unit="mm")), | ||
strip.text.y = element_text(angle=0) | ||
# text = element_text(size=7), | ||
# axis.title = element_text(size=7), | ||
# plot.title = element_text(size=7, face="bold", margin=margin(t=2, 0)), | ||
# axis.text.y = element_text(size=5), | ||
# panel.spacing = unit(.2, "mm"), | ||
# strip.text = element_text(size=12, margin=margin(t=0,r=1,b=0,l=1)), | ||
# strip.background = element_rect(size = .3), | ||
# plot.margin = margin(0.2) | ||
) | ||
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return(list(plt, potential_escapees, allele_designation_df_byt21)) | ||
} | ||
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c(allelic_plt, potential_escapees, allele_designation_df) %<-% make_allelic_plot(chr21_allelic_counts_df) |
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