Intro to R
Workshop on Plotting in R
Lokesh Mano • 18-Jan-2022
R is a programming language for statistical computing, and data wrangling. It is open-source, widely used in data science, has a wide range of functions and algorithms for graphing and data analyses.
We will use R in this course for generating plots from different biological data that are of higher quality and standard for publications. To do this, you will brush-up your memory on some important aspects of R that are important for this course below:
Before starting with the lab sessions of the entire course, you must have downloaded all the necessary files from here and make sure that the directory tree looks similar to the one displayed in that page
In that case, you can proceed with the exercise now and remember to have fun :)
1 Input/Output
Input and output of data and images is an important aspect with data analysis.
1.1 Text
Data can come in a variety of formats which needs to be read into R and converted to an R data type.
Text files are the most commonly used input. Text files can be read in using the function read.table. We have a sample file to use: iris.txt.
dfr <- read.table("data/metadata.csv",sep = ";", header=TRUE,stringsAsFactors=F)This reads in a tab-delimited text file with a header. The argument sep='\t' is set by default to specify that the delimiter is a tab. stringsAsFactors=F setting ensures that character columns are not automatically converted to factors.
It’s always a good idea to check the data after import.
head(dfr)output
## Sample_ID Sample_Name Time Replicate Cell
## 1 Sample_1 t0_A t0 A A431
## 2 Sample_2 t0_B t0 B A431
## 3 Sample_3 t0_C t0 C A431
## 4 Sample_4 t2_A t2 A A431
## 5 Sample_5 t2_B t2 B A431
## 6 Sample_6 t2_C t2 C A431str(dfr)output
## 'data.frame': 12 obs. of 5 variables:
## $ Sample_ID : chr "Sample_1" "Sample_2" "Sample_3" "Sample_4" ...
## $ Sample_Name: chr "t0_A" "t0_B" "t0_C" "t2_A" ...
## $ Time : chr "t0" "t0" "t0" "t2" ...
## $ Replicate : chr "A" "B" "C" "A" ...
## $ Cell : chr "A431" "A431" "A431" "A431" ...Check ?read.table for other wrapper functions to read in text files.
Let’s filter this data.frame and create a new data set.
dfr1 <- dfr[dfr$Time == "t0",]And we can write this as a text file.
write.table(dfr1,"iris-setosa.txt",sep="\t",row.names=F,quote=F)sep="\t" sets the delimiter to tab. row.names=F denotes that rownames should not be written. quote=F specifies that doubles must not be placed around strings.
2 Data-Frames
You have probably learnt about data.frame in your previous course and this is the most important data structure for generating plots using ggplot. So, below you have some quick exercises on how to work with Data-Frames.
Vectors positions can be accessed using []. R follows 1-based indexing, meaning that the indexing starts at 1.
Data-frame or matrix positions can be accessed using [] specifying row and column like [row,column].
dfr <- data.frame(x = 1:3, y = c("a", "b", "c"))
dfr
dfr[1,]
dfr[,1]
dfr[2,2]output
## x y
## 1 1 a
## 2 2 b
## 3 3 c
## x y
## 1 1 a
## [1] 1 2 3
## [1] "b"The function cbind() is used to join two data-frames column-wise.
dfr1 <- data.frame(x = 1:3, y = c("a", "b", "c"))
dfr2 <- data.frame(p = 4:6, q = c("d", "e", "f"))
dfr1
dfr2
cbind(dfr1,dfr2)output
## x y
## 1 1 a
## 2 2 b
## 3 3 c
## p q
## 1 4 d
## 2 5 e
## 3 6 f
## x y p q
## 1 1 a 4 d
## 2 2 b 5 e
## 3 3 c 6 fSimilarly, rbind() is used to join two data-frames row-wise.
dfr1 <- data.frame(x = 1:3, y = c("a", "b", "c"))
dfr2 <- data.frame(x = 4:6, y = c("d", "e", "f"))
dfr1
dfr2
rbind(dfr1,dfr2)output
## x y
## 1 1 a
## 2 2 b
## 3 3 c
## x y
## 1 4 d
## 2 5 e
## 3 6 f
## x y
## 1 1 a
## 2 2 b
## 3 3 c
## 4 4 d
## 5 5 e
## 6 6 fTwo data-frames can be merged based on a shared column using the merge() function.
dfr1 <- data.frame(x = 1:4, p = c("a", "b", "c","d"))
dfr2 <- data.frame(x = 3:6, q = c("l", "m", "n","o"))
dfr1
dfr2
merge(dfr1,dfr2,by="x")
merge(dfr1,dfr2,by="x",all.x=T)
merge(dfr1,dfr2,by="x",all.y=T)
merge(dfr1,dfr2,by="x",all=T)output
## x p
## 1 1 a
## 2 2 b
## 3 3 c
## 4 4 d
## x q
## 1 3 l
## 2 4 m
## 3 5 n
## 4 6 o
## x p q
## 1 3 c l
## 2 4 d m
## x p q
## 1 1 a <NA>
## 2 2 b <NA>
## 3 3 c l
## 4 4 d m
## x p q
## 1 3 c l
## 2 4 d m
## 3 5 <NA> n
## 4 6 <NA> o
## x p q
## 1 1 a <NA>
## 2 2 b <NA>
## 3 3 c l
## 4 4 d m
## 5 5 <NA> n
## 6 6 <NA> o3 Data formats
In terms of the R (and other similar programming languages), the data can be viewed or stored in two main formats! They are called wide and long formats. Below you will see what exactly they stand for and why is it important for plotting in ggplot.
3.1 Wide format
A quick preview:
Counts Table
| Sample_1 | Sample_2 | Sample_3 | Sample_4 | |
|---|---|---|---|---|
| ENSG00000000003 | 321 | 303 | 204 | 492 |
| ENSG00000000005 | 0 | 0 | 0 | 0 |
| ENSG00000000419 | 696 | 660 | 472 | 951 |
| ENSG00000000457 | 59 | 54 | 44 | 109 |
| ENSG00000000460 | 399 | 405 | 236 | 445 |
| ENSG00000000938 | 0 | 0 | 0 | 0 |
And we usually have our metadata related to the samples in another table like below:
Metadata Table
| Sample_ID | Sample_Name | Time | Replicate | Cell |
|---|---|---|---|---|
| Sample_1 | t0_A | t0 | A | A431 |
| Sample_2 | t0_B | t0 | B | A431 |
| Sample_3 | t0_C | t0 | C | A431 |
| Sample_4 | t2_A | t2 | A | A431 |
- Wide format data is called “wide” because it typically has a lot of columns which stretch widely across the page or your computer screen.
- Most of us are familiar with looking at wide format data
- It is convenient and we are more used to looking at data this way in our Excel sheets.
- It often lets you see more of the data, at one time, on your screen
3.2 Long format
Below is glimpse how the long format of the same data look like:
| Gene | Samples | count |
|---|---|---|
| ENSG00000000003 | Sample_1 | 321 |
| ENSG00000000005 | Sample_1 | 0 |
| ENSG00000000419 | Sample_1 | 696 |
| ENSG00000000457 | Sample_1 | 59 |
| ENSG00000000460 | Sample_1 | 399 |
| ENSG00000000938 | Sample_1 | 0 |
| ENSG00000000003 | Sample_2 | 303 |
| ENSG00000000005 | Sample_2 | 0 |
| ENSG00000000419 | Sample_2 | 660 |
| ENSG00000000457 | Sample_2 | 54 |
Or to be even more complete and precise:
| Sample_ID | Sample_Name | Time | Replicate | Cell | Gene | count |
|---|---|---|---|---|---|---|
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000003 | 321 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000005 | 0 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000419 | 696 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000457 | 59 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000460 | 399 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000938 | 0 |
| Sample_2 | t0_B | t0 | B | A431 | ENSG00000000003 | 303 |
| Sample_2 | t0_B | t0 | B | A431 | ENSG00000000005 | 0 |
| Sample_2 | t0_B | t0 | B | A431 | ENSG00000000419 | 660 |
| Sample_2 | t0_B | t0 | B | A431 | ENSG00000000457 | 54 |
- Long format data is typically less familiar to most humans
- It seems awfully hard to get a good look at all (or most) of it
- It seems like it would require more storage on your hard disk
- It seems like it would be harder to enter data in a long format
3.3 Which is better?
- Well, there are some contexts where putting things in wide format is computationally efficient because you can treat data in a matrix format and to efficient matrix calculations on it.
- However, adding data to wide format data sets is very hard:
- It is very difficult to conceive of analytic schemes that apply generally across all wide-format data sets.
- Many tools in R want data in long format like ggplot
- The long format for data corresponds to the relational model for storing data, which is the model used in most modern data bases like the SQL family of data base systems.
- A more technical treatment of wide versus long data requires some terminology:
- Identifier variables are often categorical things that cross-classify observations into categories.
- Measured variables are the names given to properties or characteristics that you can go out and measure.
- Values are the values that you measure are record for any particular measured variable.
- In any particular data set, what you might want to call an Identifier variables versus a Measured variables can not always be entirely clear.
- Other people might choose to define things differently.
- However, to my mind, it is less important to be able to precisely recognize these three entities in every possible situation (where there might be some fuzziness about which is which)
- And it is more important to understand how Identifier variables, Measured variables, and Values interact and behave when we are transforming data between wide and long formats.
4 Conversion between formats
As for the biological data analysis, to be able to use tools such as ggplot, in simple terms we should learn to convert our data:
As per our previous examples: We should learn to convert
From this format
| Sample_1 | Sample_2 | Sample_3 | Sample_4 | |
|---|---|---|---|---|
| ENSG00000000003 | 321 | 303 | 204 | 492 |
| ENSG00000000005 | 0 | 0 | 0 | 0 |
| ENSG00000000419 | 696 | 660 | 472 | 951 |
| ENSG00000000457 | 59 | 54 | 44 | 109 |
| ENSG00000000460 | 399 | 405 | 236 | 445 |
| ENSG00000000938 | 0 | 0 | 0 | 0 |
To this format
| Sample_ID | Sample_Name | Time | Replicate | Cell | Gene | count |
|---|---|---|---|---|---|---|
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000003 | 321 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000005 | 0 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000419 | 696 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000457 | 59 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000460 | 399 |
| Sample_1 | t0_A | t0 | A | A431 | ENSG00000000938 | 0 |
| Sample_2 | t0_B | t0 | B | A431 | ENSG00000000003 | 303 |
| Sample_2 | t0_B | t0 | B | A431 | ENSG00000000005 | 0 |
| Sample_2 | t0_B | t0 | B | A431 | ENSG00000000419 | 660 |
| Sample_2 | t0_B | t0 | B | A431 | ENSG00000000457 | 54 |
Here we will only cover the conversion from wide to long, as this is more relevant to us. For the other way around, one can look into spread() from the tidyr package.
4.1 Using reshape2
By using the melt() function from the reshape2 package we can convert the wide-formatted data into long-formatted data! Here, to combine the metadata table to the gene counts table, we will also use the merge() function like we did before!
library(reshape2)
gc <- read.table("data/counts_raw.txt", header = T, row.names = 1, sep = "\t")
md <- read.table("data/metadata.csv", header = T, sep = ";")
rownames(md) <- md$Sample_ID
#merging gene counts table with metadata
merged_data_wide <- merge(md, t(gc), by = 0)
#removing redundant columns
merged_data_wide$Row.names <- NULL
merged_data_long <- melt(merged_data_wide, id.vars = c("Sample_ID","Sample_Name","Time","Replicate","Cell"), variable.name = "Gene", value.name = "count")
head(merged_data_long)output
## Sample_ID Sample_Name Time Replicate Cell Gene count
## 1 Sample_1 t0_A t0 A A431 ENSG00000000003 321
## 2 Sample_10 t24_A t24 A A431 ENSG00000000003 950
## 3 Sample_11 t24_B t24 B A431 ENSG00000000003 760
## 4 Sample_12 t24_C t24 C A431 ENSG00000000003 1436
## 5 Sample_2 t0_B t0 B A431 ENSG00000000003 303
## 6 Sample_3 t0_C t0 C A431 ENSG00000000003 2044.2 Using tidyr
If you are more familiar with using tidyverse or tidyr packages, you can combine tables by join() and then use gather() to make long formatted data in the same command. This is a powerful and more cleaner way of dealing with data in R.
library(tidyverse)
gc_long <- gc %>%
rownames_to_column(var = "Gene") %>%
gather(Sample_ID, count, -Gene) %>%
full_join(md, by = "Sample_ID") %>%
select(Sample_ID, everything()) %>%
select(-c(Gene,count), c(Gene,count))
gc_long %>%
head(10)output
## Sample_ID Sample_Name Time Replicate Cell Gene count
## 1 Sample_1 t0_A t0 A A431 ENSG00000000003 321
## 2 Sample_1 t0_A t0 A A431 ENSG00000000005 0
## 3 Sample_1 t0_A t0 A A431 ENSG00000000419 696
## 4 Sample_1 t0_A t0 A A431 ENSG00000000457 59
## 5 Sample_1 t0_A t0 A A431 ENSG00000000460 399
## 6 Sample_1 t0_A t0 A A431 ENSG00000000938 0
## 7 Sample_1 t0_A t0 A A431 ENSG00000000971 0
## 8 Sample_1 t0_A t0 A A431 ENSG00000001036 844
## 9 Sample_1 t0_A t0 A A431 ENSG00000001084 535
## 10 Sample_1 t0_A t0 A A431 ENSG00000001167 4935 Exercise
Task Here in this exercise we have used the counts_raw.txt file, you can try to make similar R objects for each of the other three counts (counts_filtered.txt, counts_vst.txt and counts_deseq2.txt) in long format. So for example you would have gc_filt, gc_vst and gc_deseq2 R objects in the end.
Tip Remember to take a look at how these files are formatted, before you import!
6 Acknowledgements
Much of the data format explanations and exercises were obtained from the GitHub of Eric C. Anderson
7 Getting help
- Use
?functionto get function documentation - Use
??blato search for a function - Use
args(function)to get the arguments to a function - Go to the package CRAN page/webpage for vignettes
- R Cookbook: General purpose reference.
- Quick R: General purpose reference.
- Stackoverflow: Online community to find solutions to your problems.
8 Session info
sessionInfo()## R version 4.1.2 (2021-11-01)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.6 LTS
##
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/openblas/libblas.so.3
## LAPACK: /usr/lib/x86_64-linux-gnu/libopenblasp-r0.2.20.so
##
## locale:
## [1] LC_CTYPE=C.UTF-8 LC_NUMERIC=C LC_TIME=C.UTF-8
## [4] LC_COLLATE=C.UTF-8 LC_MONETARY=C.UTF-8 LC_MESSAGES=C.UTF-8
## [7] LC_PAPER=C.UTF-8 LC_NAME=C LC_ADDRESS=C
## [10] LC_TELEPHONE=C LC_MEASUREMENT=C.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] ggimage_0.3.0 treeio_1.18.1 ggtree_3.2.1
## [4] pheatmap_1.0.12 leaflet_2.0.4.1 swemaps_1.0
## [7] mapdata_2.3.0 maps_3.4.0 ggpubr_0.4.0
## [10] cowplot_1.1.1 ggthemes_4.2.4 scales_1.1.1
## [13] ggrepel_0.9.1 wesanderson_0.3.6 forcats_0.5.1
## [16] stringr_1.4.0 purrr_0.3.4 readr_2.1.1
## [19] tidyr_1.1.4 tibble_3.1.6 tidyverse_1.3.1
## [22] reshape2_1.4.4 ggplot2_3.3.5 formattable_0.2.1
## [25] kableExtra_1.3.4 dplyr_1.0.7 lubridate_1.8.0
## [28] yaml_2.2.1 fontawesome_0.2.2.9000 captioner_2.2.3
## [31] bookdown_0.24 knitr_1.37
##
## loaded via a namespace (and not attached):
## [1] colorspace_2.0-2 ggsignif_0.6.3 ellipsis_0.3.2
## [4] fs_1.5.2 aplot_0.1.2 rstudioapi_0.13
## [7] farver_2.1.0 fansi_1.0.2 xml2_1.3.3
## [10] splines_4.1.2 jsonlite_1.7.3 broom_0.7.11
## [13] dbplyr_2.1.1 compiler_4.1.2 httr_1.4.2
## [16] backports_1.4.1 assertthat_0.2.1 Matrix_1.3-4
## [19] fastmap_1.1.0 lazyeval_0.2.2 cli_3.1.0
## [22] leaflet.providers_1.9.0 htmltools_0.5.2 tools_4.1.2
## [25] gtable_0.3.0 glue_1.6.0 Rcpp_1.0.8
## [28] carData_3.0-5 cellranger_1.1.0 jquerylib_0.1.4
## [31] vctrs_0.3.8 ape_5.6-1 svglite_2.0.0
## [34] nlme_3.1-153 crosstalk_1.2.0 xfun_0.29
## [37] rvest_1.0.2 lifecycle_1.0.1 rstatix_0.7.0
## [40] hms_1.1.1 parallel_4.1.2 RColorBrewer_1.1-2
## [43] curl_4.3.2 gridExtra_2.3 ggfun_0.0.4
## [46] yulab.utils_0.0.4 sass_0.4.0 stringi_1.7.6
## [49] highr_0.9 tidytree_0.3.7 rlang_0.4.12
## [52] pkgconfig_2.0.3 systemfonts_1.0.3 evaluate_0.14
## [55] lattice_0.20-45 patchwork_1.1.1 htmlwidgets_1.5.4
## [58] labeling_0.4.2 tidyselect_1.1.1 plyr_1.8.6
## [61] magrittr_2.0.1 R6_2.5.1 magick_2.7.3
## [64] generics_0.1.1 DBI_1.1.2 pillar_1.6.4
## [67] haven_2.4.3 withr_2.4.3 mgcv_1.8-38
## [70] abind_1.4-5 modelr_0.1.8 crayon_1.4.2
## [73] car_3.0-12 utf8_1.2.2 tzdb_0.2.0
## [76] rmarkdown_2.11 grid_4.1.2 readxl_1.3.1
## [79] reprex_2.0.1 digest_0.6.29 webshot_0.5.2
## [82] gridGraphics_0.5-1 munsell_0.5.0 viridisLite_0.4.0
## [85] ggplotify_0.1.0 bslib_0.3.1