Introduction to dotplots in R
Nathan Brouwer
10/21/2021
Sequence dotplots in R
By: Avril Coghlan.
Adapted, edited and expanded: Nathan Brouwer under the Creative Commons 3.0 Attribution License (CC BY 3.0).
NOTE: I’ve added some new material that is rather terse and lacks explication.
Good sources of more info: https://omicstutorials.com/interpreting-dot-plot-bioinformatics-with-an-example/
As a first step in comparing two protein, RNA or DNA sequences, it is a good idea to make a dotplot. A dotplot is a graphical method that allows the comparison of two protein or DNA sequences and identify regions of close similarity between them. A dotplot is essentially a two-dimensional matrix (like a grid), which has the sequences of the proteins being compared along the vertical and horizontal axes.
In order to make a simple dotplot to represent of the similarity between two sequences, individual cells in the matrix can be shaded black if residues are identical, so that matching sequence segments appear as runs of diagonal lines across the matrix. Identical proteins will have a line exactly on the main diagonal of the dotplot, that spans across the whole matrix.
For proteins that are not identical, but share regions of similarity, the dotplot will have shorter lines that may be on the main diagonal, or off the main diagonal of the matrix. In essence, a dotplot will reveal if there are any regions that are clearly very similar in two protein (or DNA) sequences.
Preliminaries
library(compbio4all)
library(rentrez)Visualzing two identical sequences
To help build our intuition about dotplots we’ll first look at some artificial examples. First, we’ll see what happens when we make a dotplot comparing the alphabet versus itself. The build-in LETTERS object in R contains the alphabet from A to Z. This is a sequence with no repeats.
#LETTERS
seqinr::dotPlot(LETTERS , LETTERS ) # add code
What we get is a perfect diagonal line.
Visualizing repeats
This chunk of code is creating new vector consisting of the alphabet two times and then making a dotplot of the new vector against itself
LETTERS.2.times <- c(LETTERS, LETTERS)
seqinr::dotPlot(LETTERS.2.times,
LETTERS.2.times)
This code chunk is doing the same thing as the earlier one but the vector consists of the alphabet 3 times.
LETTERS.3.times <- c(LETTERS, LETTERS, LETTERS)
seqinr::dotPlot(LETTERS.3.times, LETTERS.3.times)
These chunks of code create a once repeated sequence of A through I and then creates a dotplot of the sequencde against itself
seq.repeat <- c("A","C","D","E","F","G","H","I")
seq.repeat2times <- c(seq.repeat, seq.repeat)Make the dotplot:
seqinr::dotPlot(seq.repeat2times, seq.repeat2times)
Dotplot with inversion
This code chunk is inverting the alphabet sequence listed 3 times back to back and then creating a dotplot of it against itself.
“invert” means “inversion”
LETTERS.3.times.with.invert <- rev(LETTERS.3.times)
seqinr::dotPlot(LETTERS.3.times.with.invert, LETTERS.3.times.with.invert)# add code
This code chunk is creating 3 segments of the alphabet and a new vector is being created where letters 17-16 are placed in between letters 1-8 and 9-16, so “translocation” has occurred. After, a dotplot is created with the sequence against itself.
seg1 <- LETTERS[1:8]
seg2 <- LETTERS[9:16]
seg3 <- LETTERS[17:26]
LETTERS.with.transloc <- c(seg1, seg3, seg2)
seqinr::dotPlot(LETTERS.with.transloc, LETTERS.with.transloc)
LETTERS sequence with sample()
This code chunk is taking a sample of out of LETTERS 26 times and creates a “sequence” of each selected letter.
sample(x = LETTERS, size = 26, replace = T)## [1] "O" "N" "G" "G" "T" "K" "C" "Y" "P" "D" "Q" "N" "U" "T" "N" "E" "E" "O" "C"
## [20] "D" "L" "Y" "A" "A" "P" "H"letters.rand1 <- sample(x = LETTERS, size = 26, replace = F)
letters.rand2 <- sample(x = LETTERS, size = 26, replace = T)
seqinr::dotPlot(letters.rand1, letters.rand2
)
Download sequences
Now we’ll make a real dotplot of the chorismate lyase proteins from two closely related species, Mycobacterium leprae and Mycobacterium ulcerans.
Note - these are protein sequences so db = “protein”
TODO: briefly summarize these steps
# sequence 1: Q9CD83
leprae_fasta <- rentrez::entrez_fetch(db = "protein",
id = "Q9CD83",
rettype = "fasta")
# sequence 2: OIN17619.1
ulcerans_fasta <- rentrez::entrez_fetch(db ="protein",
id = "OIN17619.1",
rettype = "fasta")
# add code
leprae_vector <- compbio4all::fasta_cleaner(leprae_fasta)
ulcerans_vector <- compbio4all::fasta_cleaner(ulcerans_fasta)Creating a dotplot
We can create a dotplot for two sequences using the dotPlot() function in the seqinr package.
First, let’s look at a dotplot created using only a single sequence. This is frequently done to investigate a sequence for the presence of repeats.
(Note - and older version of this exercise stated this kind of anlysis wasn’t normally done; this was written last year before I knew of the use of dotplots for investigating sequence repeats.)
seqinr::dotPlot(leprae_vector, ulcerans_vector)
TODO: The dotplot shows the identical amino acids in the two sequences, and the diagonal line indicates that there exist several identical amino acids at similar positions.
In the dotplot above, the M. leprae sequence is plotted along the x-axis (horizontal axis), and the M. ulcerans sequence is plotted along the y-axis (vertical axis). The dotplot displays a dot at points where there is an identical amino acid in the two sequences.
For example, if amino acid 53 in the M. leprae sequence is the same amino acid (eg. “W”) as amino acid 70 in the M. ulcerans sequence, then the dotplot will show a dot the position in the plot where x =50 and y =53.
In this case you can see a lot of dots along a diagonal line, which indicates that the two protein sequences contain many identical amino acids at the same (or very similar) positions along their lengths. This is what you would expect, because we know that these two proteins are homologs (related proteins) because they share a close evolutionary history.