article_figures.Rmd 14.5 KB
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---
title: "article_figures.Rmd"
author: "Marie Bourdon"
date: "July 2021"
output: html_document
---

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# Goal and raw data
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The goal of this script is to produce figure for the stuart package manuscript.

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This scripts uses data from the package, and other files found in the "files" directory.
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This script uses the qtl_plot() ggplot based function to plot QTL mapping results. This function is in the script "QTL_plot.R".

```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = TRUE)
library(tidyverse)
library(magrittr)
library(qtl)
library(cowplot)
library(grid)
library(gridExtra)
library(gridGraphics)

library(stuart)

source("files/QTL_plot.R")
```

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# Data load and use of stuart functions
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## genos dataframe
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Data frame from stuart with genotypes of 176 F2 individuals and parental strains.
```{r genos}
data(genos)
summary(genos)
```
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## phenos dataframe
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Data frame from stuart with phenotypes of 176 F2 individuals for a quantitative trait.
```{r phenos}
data(phenos)
summary(phenos)
```
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## annotation file
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Annotation file from K.Broman: https://kbroman.org/MUGAarrays/mini_annotations.html
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```{r annot}
annot_mini <- read.csv(url("https://raw.githubusercontent.com/kbroman/MUGAarrays/master/UWisc/mini_uwisc_v2.csv"))
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summary(annot_mini)
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```
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## parental strains genotypes
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2 genotypes tables will be used:

* One table with our genotypes of the strains used in the cross: "strains"

* One table with reference genotypes: "strns_ref"
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```{r strains}
#our genotypes
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#create tibble with individivual names
parental_strains <- tibble::tibble(parent1 = c("StrainsA_1","StrainsA_2"),
                                   parent2 = c("StrainsB_1","StrainsB_2"))


#create data frame with geno_strains
strains <- geno_strains(annot=annot_mini,geno=genos,
                        strn=parental_strains,cols=c("chr","cM_cox"))
rm(parental_strains)

#remove genotypes of parental strains from genos data frame
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genos %<>% filter(!Sample.ID %in% c("StrainsA_1","StrainsA_2","StrainsB_1","StrainsB_2"))

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#summary
summary(strains)
```

```{r strns_ref}
#reference genotypes
#load parental strains genotype data from Neogen
strns_ref <- read.csv("files/ref_genotypes.csv")

#join with annotation file from miniMUGA
strns_ref <- strns_ref %>% select(name,parent1,parent2) %>% right_join(annot_mini,.,by=c("marker"="name")) %>% select(c(marker,chr,cM_cox,parent1,parent2))

#summary
summary(strns_ref)
```

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## Use of stuart's functions
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```{r}
data(stuart_tab)
summary(stuart_tab)

tab2 <- mark_match(stuart_tab,ref=strns_ref)
tab2 <- mark_poly(tab2)
tab2 <- mark_prop(tab2,cross="F2",homo=0.1,hetero=0.1,homo1X=c(0.1,1),homo2X=c(0.1,1),heteroX=c(0.1,1))
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tab2 <- mark_allele(tab2,ref=strns_ref,cross="F2",par1="parent1",par2="parent2")
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data(stuart_newmap)
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tab2 <- mark_estmap(tab=tab2,map=stuart_newmap,annot=annot_mini)
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```

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# Graphs

## Grid mark_prop

### Graph missing genotypes

```{r graph_NA}
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na_plot <- tab2 %>% mutate(prop_NA=n_NA/176) %>% ggplot(aes(x=prop_NA)) +
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  geom_histogram() +
  scale_y_log10() +
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  theme_classic() +
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  labs(title="Proportion of missing genotyped",
       x="Proportion of NA",y="Number of markers") +
  theme(
    aspect.ratio=0.8,
    plot.title = element_text(hjust = 0.4,face="bold",size=14))

na_plot
```

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Proportions of markers with more than 75% of missing genotypes:
```{r prop_missing}
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tab2 %>% mutate(prop_NA=n_NA/176) %>% filter(prop_NA > 0.50) %>% summarise(p=n()/count(tab2)%>%pull())
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```


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### Graph proportion of genotypes

```{r graph_prop}
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prop_plot <- tab2 %>% filter(n_NA<88) %>% filter(!chr %in% c("M","X","Y")) %>%
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  ggplot(aes(x=n_HM1/(n_HM1+n_HM2+n_HT),y=n_HM2/(n_HM1+n_HM2+n_HT),color=as.factor(exclude_prop))) +
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  geom_point() +
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  scale_color_manual(values=c("#66bd63","#b2182b"),labels = c("Retained", "Excluded")) +
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  geom_hline(yintercept = 0.1,linetype="dashed",size=.3) +
  geom_vline(xintercept = 0.1,linetype="dashed",size=.3) +
  geom_abline(intercept = 0.9, slope=-1,linetype="dashed",size=.3) +
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  #geom_text(aes(label=ifelse(exclude_prop=="1",SNP.Name,'')),hjust=0, vjust=0,size=2) +
  labs(title="Exclusion of markers with mark_prop()",
       x="Proportion of homozygous individuals AA",
       y="Proportion of homozygous individuals BB",
       color="Exclusion") +
  theme_classic() +
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  theme(aspect.ratio=0.8,
        legend.position=c(0.8,0.8),
        legend.title = element_blank()) +
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  theme(plot.title = element_text(hjust = 0.4,face="bold",size=14)) +
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  geom_segment(x = 0.2, y = 0.78,
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               xend = 0.25, yend = 0.72,
               color = "#1d91c0",
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               arrow = arrow(type="closed",length=unit(4,"points"))) +
  geom_segment(x = 0.39, y = 0.58,
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               xend = 0.44, yend = 0.52,
               color = "#1d91c0",
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               arrow = arrow(type="closed",length=unit(4,"points")))
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prop_plot
```
### Grid

Figure with graphs proportion of NA and exclusion depending on genotype proportions


```{r grid_na_prop}
grid <- plot_grid(na_plot,prop_plot,
                  ncol=1,
                  labels = c('A', 'B'), 
                  label_size = 20,
                  byrow = TRUE
                  )

grid

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ggsave("figures/fig2.pdf",grid,width=5,height=8)
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ggsave("figures/fig2.png",grid,width=5,height=8)
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rm(na_plot,prop_plot)
```

## Graph before after

### Before: creation of Rqtl csv file 

```{r cross_before}
# filter at minima: remove non polymorphic and NA>0.5 
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tab_before <- mark_na(stuart_tab)
tab_before <- mark_poly(tab_before)
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# create rqtl csv file
write_rqtl(geno=genos,pheno=phenos,tab=tab_before,ref=strns_ref,par1="parent1",par2="parent2",prefix="ind_",pos="cM_cox",path="files/cluster/cross_before.csv")
```

### Before: newmap and permutation

These objects were produced on our cluster with the following script: /files/cluster/before_after.R

### Before: plot estimated map

```{r before_map}
# import cross
cross_before <- read.cross(format="csv",file="files/cluster/cross_before.csv",
                              genotypes=c("0","1","2"),na.strings=c("NA"), convertXdata=TRUE)

# load rda with newmap
load("files/cluster/newmap_before.rda")

# plot
plotMap(cross_before,newmap_before,shift=TRUE)
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plotmap_before <- ~plotMap(cross_before,newmap_before,shift=TRUE,main="Before stuart", ylab='')
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```

### Before: plot genome scan

```{r before_scan}
# load rda with perm
load("files/cluster/before_1000p.rda")
# load("files/before_1000p.rda")


# calculate thresholds
threshold_before <- summary(before_1000p,alpha=c(0.05,0.1,0.63)) #donne lod score pour risque 0.05, 0.1, 0.63

# scanone
cross_before <- calc.genoprob(cross_before, step=2.0, off.end=0.0, 
                                 error.prob=1.0E-4, map.function="haldane", stepwidth="fixed")


pheno_before <- scanone(cross=cross_before, chr=c("1", "2", "3", "4", "5", "6", "7", "8", "9", "10", "11", "12", "13", "14", "15", "16", "17", "18", "19", "X", "Y"), pheno.col="Pheno", model="normal", method="em")
summary(pheno_before)

# Plot
pheno_before_plot <- qtl_plot(pheno_before,lod=data.frame(group = c("alpha=0.05", "alpha=0.1","alpha=0.63"),
                 lod = threshold_before[1:3]),
         ylab="LOD score",
         title="QTL mapping",
         x.label = element_blank(),
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         size=0.6,
         strip.pos="bottom") +
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    theme(legend.position = "none") +
    ggtitle("")
pheno_before_plot
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```

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```{r before_scan}
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pheno_before_zoom <- qtl_plot(pheno_before,lod=data.frame(group = c("alpha=0.05", "alpha=0.1","alpha=0.63"),
                 lod = threshold_before[1:3]),
         ylab="LOD score",
         chrs = "13",
         size=0.6) +
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    theme(legend.position = "none",
          strip.background = element_blank(),
          strip.text.x = element_blank()) +
    xlab("Position (cM)") +
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    ggtitle("")
pheno_before_zoom
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```

### Before figure
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```{r grid_before}
grid_before <- plot_grid(as_grob(plotmap_before),pheno_before_plot,pheno_before_zoom,ncol=1,labels=c("A","B","C"),label_size=20)

ggsave("figures/fig1.pdf",grid_before,width=7,height = 15)
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ggsave("figures/fig1.png",grid_before,width=7,height = 15)
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```


### After: creation of Rqtl csv file 

```{r cross_after}
# filter with stuart functions: use the good data for parental strains (strains df)
tab2 <- mark_match(stuart_tab,ref=strains)
tab2 <- mark_poly(tab2)
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tab2 <- mark_na(tab2)
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tab2 <- mark_prop(tab2,cross="F2",homo=0.1,hetero=0.1,homo1X=c(0.1,1),homo2X=c(0.1,1),heteroX=c(0.1,1))
tab2 <- mark_allele(tab2,ref=strains,cross="F2",par1="parent1",par2="parent2")


# create rqtl csv file
write_rqtl(geno=genos,pheno=phenos,tab=tab2,ref=strains,par1="parent1",par2="parent2",prefix="ind_",pos="cM_cox",path="files/cluster/cross_after.csv")
```

### After: newmap 

These objects were produced on our cluster with the following script: /files/cluster/before_after.R

### After: plot estimated map

```{r after_map}
# import cross
cross_after <- read.cross(format="csv",file="files/cluster/cross_after.csv",
                              genotypes=c("0","1","2"),na.strings=c("NA"), convertXdata=TRUE)

# load rda with newmap
load("files/cluster/newmap_after.rda")

# plot
plotMap(cross_after,newmap_after,shift=TRUE)
plotmap_after <- ~plotMap(cross_after,newmap_after,shift=TRUE,main="After stuart")
```
### Remove last problematic markers with mark_estmap

```{r after_estmap}
#filter with mark_estmap
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tab2 <- mark_estmap(tab2,newmap_after,annot_mini)
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# create rqtl csv file
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write_rqtl(geno=genos,pheno=phenos,tab=tab2,ref=strains,par1="parent1",par2="parent2",prefix="ind_",pos="cM_cox",path="files/cluster2/cross_after2.csv")
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```

### After: plot estimated map 2

```{r after_map2}
# import cross
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cross_after2 <- read.cross(format="csv",file="files/cluster2/cross_after2.csv",
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                              genotypes=c("0","1","2"),na.strings=c("NA"), convertXdata=TRUE)

# load rda with newmap
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load("files/cluster2/newmap_after2.rda")
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# plot
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plotMap(cross_after2,newmap_after2,shift=TRUE)
plotmap_after <- ~plotMap(cross_after2,newmap_after2,shift=TRUE,main="After stuart")
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```

### After: plot genome scan

```{r after_scan}
# load rda with perm
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load("files/cluster2/after_1000p2.rda")
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# calculate thresholds
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threshold_after <- summary(after_1000p2,alpha=c(0.05,0.1,0.63)) #donne lod score pour risque 0.05, 0.1, 0.63
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# scanone
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cross_after <- calc.genoprob(cross_after2, step=2.0, off.end=0.0, 
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                                 error.prob=1.0E-4, map.function="haldane", stepwidth="fixed")


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pheno_after <- scanone(cross=cross_after2, chr=c("1", "2", "3", "4", "5", "6", "7", "8", "9", "10", "11", "12", "13", "14", "15", "16", "17", "18", "19", "X", "Y"), pheno.col="Pheno", model="normal", method="em")
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summary(pheno_after)

# Plot
pheno_after_plot <- qtl_plot(pheno_after,lod=data.frame(group = c("alpha=0.05", "alpha=0.1","alpha=0.63"),
                 lod = threshold_after[1:3]),
         ylab="LOD score",
         title="QTL mapping",
         x.label = element_blank(),
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         size=0.6,
         strip.pos="bottom") +
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    theme(legend.position = "none") +
    ggtitle("")
pheno_after_plot
```
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### Grid after
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```{r grid fig1, fig.height = 7, fig.width = 13, fig.align = "center"}
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grid_after <- plot_grid(as_grob(plotmap_after),pheno_after_plot,ncol=1,labels=c("A","B"),label_size=20)
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ggsave("figures/fig3.pdf",grid_after,width=7,height = 10)
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ggsave("figures/fig3.png",grid_after,width=7,height = 10)
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```

# Differences between real and reference genotypes for parental strains

```{r dif_number}
dif <- full_join(strains,strns_ref,by=c("marker","chr","cM_cox")) %>% 
  mutate(dif=case_when((!parent1.x%in%c("N","H") & 
                          !parent1.y%in%c("N","H") & 
                          parent1.x!=parent1.y) ~ 1,
                       (!parent2.x%in%c("N","H") &
                          !parent2.y%in%c("N","H") & 
                          parent2.x!=parent2.y) ~ 1, 
                       T~0))

dif %>% filter(dif==1) %>% count()
```

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## Table alleles different between parental strains and F2s

```{r allele}
#investigation of the role of mark_allele function
#prove that some marker with non corresponding alleles between parents and F2

#keep only markers that are exlcuded with mark_allele
allele <- tab2%>% 
  filter(exclude_allele==1&exclude_poly==0&exclude_prop==0&exclude_na==0&exclude_estmap==0)
strains_allele <- strains %>% filter(marker %in% allele$marker)

#number of markers not excluded by other functoins
tab2 %>% 
  filter(exclude_poly==0&exclude_prop==0&exclude_na==0&exclude_estmap==0) %>% nrow()

#join with strains genotypes to have parental strains
allele <- left_join(allele,strains_allele,by=c("marker"="marker"))


#most of markers excluded with mark_allele that were not excluded with other functions have N/H as genotype for parents
#keep only those with non missing/heterozygous genotypes
allele %<>% filter(parent1 != "N" & parent2 != "N")
allele %<>% select(marker,parent1,parent2,allele_1,allele_2)


print(allele)

print(xtable::xtable(allele, type = "latex"), file = "tables/tab_alleles.tex",include.rownames=FALSE)
rm(allele,strains_allele)
```

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```{r dif_table}
table_dif <- dif %>% filter(dif==1) %>% select(marker,parent1_ref=parent1.y,parent1_geno=parent1.x,parent2_ref=parent2.y,parent2_geno=parent2.x) %>% head()
knitr::kable(table_dif)
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```

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## Number of markers kept after each function

```{r barplot}
none <- tab2 %>% nrow()
match <- tab2 %>% filter(exclude_match==0) %>% nrow()
allele <- tab2 %>% filter(exclude_match==0&exclude_allele==0) %>% nrow()
naf <- tab2 %>% filter(exclude_match==0&exclude_allele==0&exclude_na==0) %>% nrow()
poly <- tab2 %>% filter(exclude_match==0&exclude_allele==0&exclude_na==0&exclude_poly==0) %>% nrow()
prop <- tab2 %>% filter(exclude_match==0&exclude_allele==0&exclude_na==0&exclude_poly==0&exclude_prop==0) %>% nrow()
estmap <- tab2 %>% filter(exclude_match==0&exclude_allele==0&exclude_na==0&exclude_poly==0&exclude_prop==0&exclude_estmap==0) %>% nrow()

functions_df <- tibble(fct=c("none","match","allele","na","poly","prop","estmap"),
                       markers=c(none,match,allele,naf,poly,prop,estmap))

functions_plot <- functions_df %>% ggplot(aes(x=markers,y=fct)) +
  geom_bar(stat="identity",width=0.6) +
  geom_text(aes(label=markers), hjust=1.3, color="white", size=3.5) +
  scale_y_discrete(limits=c("estmap","prop","poly", "na", "allele","match","none")) +
  theme(aspect.ratio=0.7) +
  labs(title="Number of markers kept after each step",
       x="Number of markers",
       y="Function used") +
  theme_classic() +
  theme(plot.title = element_text(hjust = 0.4,face="bold",size=14))

functions_plot
rm(none,allele,match,poly,prop,barplot_df)
```

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# Pheno data format
```{r pheno}
format_pheno <- phenos[1:6,]
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write_csv(format_pheno,file="sup/tableS1.csv")
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print(xtable::xtable(format_pheno, type = "latex"), file = "tables/tab_alleles.tex",include.rownames=FALSE)
```