Analisis Relativo

install.packages("pacman")

## Installing package into '/cloud/lib/x86_64-pc-linux-gnu-library/4.4'
## (as 'lib' is unspecified)

library("pacman")

p_load( "ggplot2", 
       "dplyr", 
       "vroom")

Datos_PCR <- vroom (file = "https://raw.githubusercontent.com/ManuelLaraMVZ/resultados_PCR_practica/refs/heads/main/Cts1.csv")

## Rows: 32 Columns: 5
## ── Column specification ────────────────────────────────────────────────────────
## Delimiter: ","
## chr (4): Well, Grupo, Practica, Fluor
## dbl (1): Cq
## 
## ℹ Use `spec()` to retrieve the full column specification for this data.
## ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Datos_PCR

## # A tibble: 32 × 5
##    Well     Grupo Practica Fluor    Cq
##    <chr>    <chr> <chr>    <chr> <dbl>
##  1 A01      G1    Relativa SYBR   30.8
##  2 B01      G1    Relativa SYBR   41  
##  3 C01      G1    Relativa SYBR   26.1
##  4 D01      G1    Relativa SYBR   41  
##  5 E01      G1    Relativa SYBR   41  
##  6 F01      G1    Relativa SYBR   22.3
##  7 Profesor G1    Relativa SYBR   30.7
##  8 Profesor G2    Relativa SYBR   30.7
##  9 B01      G2    Relativa SYBR   20.3
## 10 C01      G2    Relativa SYBR   18.7
## # ℹ 22 more rows

Filtrat datos

Filtrado <- Datos_PCR %>% 
  filter(Practica == "Relativa") %>% 
  filter(Grupo == "G1") %>% 
  select( "Well", "Cq")

Filtrado

## # A tibble: 7 × 2
##   Well        Cq
##   <chr>    <dbl>
## 1 A01       30.8
## 2 B01       41  
## 3 C01       26.1
## 4 D01       41  
## 5 E01       41  
## 6 F01       22.3
## 7 Profesor  30.7

valor de gen de referencia

Referencia <- Datos_PCR %>% 
  filter (Grupo == "Referencia") %>% 
  select(Well, "Cq")

Referencia

## # A tibble: 1 × 2
##   Well          Cq
##   <chr>      <dbl>
## 1 Referencia  18.4

Valor 2^DCt

Dos_DCt <- Filtrado %>% 
  mutate(DCt = (Cq - Referencia $Cq),
         Dos_DCt = 2^-DCt)
Dos_DCt

## # A tibble: 7 × 4
##   Well        Cq   DCt     Dos_DCt
##   <chr>    <dbl> <dbl>       <dbl>
## 1 A01       30.8 12.4  0.000181   
## 2 B01       41   22.6  0.000000157
## 3 C01       26.1  7.72 0.00473    
## 4 D01       41   22.6  0.000000157
## 5 E01       41   22.6  0.000000157
## 6 F01       22.3  3.94 0.0652     
## 7 Profesor  30.7 12.3  0.000198

Obtener el valor de 2^DDCt

Dos_DDCt <- Dos_DCt %>%
  mutate(Dos_DDCt = Dos_DCt / last(Dos_DCt),
         L2 = log2(Dos_DDCt))  %>% 
  slice(4,7)
Dos_DDCt

## # A tibble: 2 × 6
##   Well        Cq   DCt     Dos_DCt Dos_DDCt    L2
##   <chr>    <dbl> <dbl>       <dbl>    <dbl> <dbl>
## 1 D01       41    22.6 0.000000157 0.000794 -10.3
## 2 Profesor  30.7  12.3 0.000198    1          0

Graficar datos

Grafica_comparativa <- ggplot(Dos_DDCt,
                              aes(x= Well,
                                  y= Dos_DDCt,
                                  fill = Well))+
  geom_col() +
  theme_classic()+
  labs(title = "Análisis relativo RT -qPCR",
       subtitle = "Tejido: testículo",
       caption = "Diseñó : Monica, Marian, Sofia y Laura",
       x= "Muestra",
       y = "Fold Change (2^-DDCt)")

Grafica_comparativa

Graficar logaritmo

Dos_DDCt2 <- Dos_DCt %>%
  mutate(Dos_DDCt = Dos_DCt / last(Dos_DCt),
         L2 = log2(Dos_DDCt))  
 
Dos_DDCt2

## # A tibble: 7 × 6
##   Well        Cq   DCt     Dos_DCt   Dos_DDCt      L2
##   <chr>    <dbl> <dbl>       <dbl>      <dbl>   <dbl>
## 1 A01       30.8 12.4  0.000181      0.915     -0.128
## 2 B01       41   22.6  0.000000157   0.000794 -10.3  
## 3 C01       26.1  7.72 0.00473      23.9        4.58 
## 4 D01       41   22.6  0.000000157   0.000794 -10.3  
## 5 E01       41   22.6  0.000000157   0.000794 -10.3  
## 6 F01       22.3  3.94 0.0652      329.         8.36 
## 7 Profesor  30.7 12.3  0.000198      1          0

Grafica_comparativa2 <- ggplot(Dos_DDCt2,
                              aes(x= Well,
                                  y= L2,
                                  fill = Well))+
  geom_col()+
  theme_classic()+
  labs(title = "Análisis relativo RT -qPCR",
       subtitle = "Tejido: testículo",
       caption = "Diseñó : Monica, Marian, Sofia y Laura",
       x= "Muestra",
       y = "log2 Fold Change")+
  geom_hline (yintercept = 0,
              liney = "solid",
              color = "black",
              linewidth = 0.2)

## Warning in geom_hline(yintercept = 0, liney = "solid", color = "black", :
## Ignoring unknown parameters: `liney`

Grafica_comparativa2