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/

http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/650/Examples_interpretations_dot_plots.html

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)
library(seqinr)

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

Creates an object that creates a grid double the size. “Think of it as have […] been divided into a 2x2 grid”. Helps show the IDENTITY LINE, as well as OFF-DIAGONAL lines.

IDENTITY LINE: 1-to-1 line of the plotted numbers

OFF-DIAGONAL LINES: Identity lines that occur in other positions on the X/Y axis

LETTERS.2.times  <- c(LETTERS, LETTERS)
length(LETTERS.2.times)
## [1] 52
seqinr::dotPlot(LETTERS.2.times, 
                LETTERS.2.times)

Shows three sequences of the LETTERS value

LETTERS.3.times <- c(LETTERS, LETTERS, LETTERS)
length(LETTERS.3.times)
## [1] 78
seqinr::dotPlot(LETTERS.3.times, LETTERS.3.times)

The rep() function takes two arguments: a vector, and the number of repeats EX. rep(a.vector, 2) <- will repeat a.vector values twice

Here we use the rep() function to repeat the seq.repeat variable

seq.repeat <- c("A","C","D","E","F","G","H","I")

# add code
seq1 <- rep(seq.repeat, 3)

Make the dotplot:

# add code
seqinr::dotPlot(seq1, seq1)

Inversing repeats

rev() function reverses a given vector sequence

“invert” means “inversion”

LETTERS.3.times.with.invert <- c(LETTERS, rev(LETTERS), LETTERS)  # add code

seqinr::dotPlot(LETTERS.3.times.with.invert, LETTERS.3.times.with.invert)# add code

Translocating Repeats

use the [] symbol following a vector to select a range of the values EX. LETTERS[1:18] selects the first 18 values LETTERS[9:18] selects the values from index 9 to 18

seg1 <- LETTERS[1:8]
seg2 <- LETTERS[9:18] # add code
seg3 <- LETTERS[18:26] # add code


LETTERS.with.transloc <- c(seg1, seg3, seg2) # add code

seqinr::dotPlot(LETTERS.with.transloc,LETTERS.with.transloc)  # add code

Generating Random Sequences

sample() selects a random sample from a vector

argument ‘replace’ dictates whether the same value can be re-selected

sample(x = LETTERS, size = 26, replace = T)
##  [1] "F" "G" "N" "B" "R" "C" "N" "J" "R" "Z" "I" "W" "L" "O" "K" "A" "T" "J" "G"
## [20] "D" "R" "V" "G" "I" "O" "Q"
letters.rand1 <- sample(x = LETTERS, size = 26, replace = F)  # add code
letters.rand2 <- sample(x = LETTERS, size = 26, replace = F)   # add code


seqinr::dotPlot(letters.rand1,letters.rand2)  # add code

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”

the dotplot aids in showing similarities between species based on their genomic data

# sequence 1: Q9CD83
leprae_fasta <- rentrez::entrez_fetch(db = "protein",    # add code
                                      id = "Q9CD83", 
                                      rettype = "fasta")
# sequence 2: OIN17619.1
ulcerans_fasta <- rentrez::entrez_fetch(db ="protein",    # add code
                                        id = "OIN17619.1", 
                                        rettype = "fasta")

# add code
leprae_vector <- compbio4all::fasta_cleaner(leprae_fasta, parse = TRUE)
ulcerans_vector <- compbio4all::fasta_cleaner(ulcerans_fasta, parse = TRUE) # add code

Using seqinr

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,      # add code
                xlab = "M. leprae", 
                ylab = "M. ulcerans")

TODO: briefly explain the main pattern

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.