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)

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

Here we are making a dot plot of the letters sequence repeated twice. This results in off diagonal lines above and below the identity line.

LETTERS.2.times  <- c(LETTERS,LETTERS)

seqinr::dotPlot(LETTERS.2.times, 
                LETTERS.2.times)

When we repeat the letters sequence three times and make a dot plot out of the resulting sequence, we end up with two sets of 2 off diagonal lines above and below the identity line. Off diagonal lines are an indicator of repeated sequence.

LETTERS.3.times <- rep(LETTERS, 3)

seqinr::dotPlot(LETTERS.3.times, 
                LETTERS.3.times)

Here we are doing the same thing as above except with a smaller set of letters.

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

seq1 <- rep(seq.repeat, 3)

Make the dotplot:

seqinr::dotPlot(seq1, 
                seq1)

Repeats with Inversions

Here, the LETTERS sequence is repeated 3 times like above, except the middle repeat is also inverted. This results in the perpendicular lines that intersect the identity line and the off diagonal lines running parallel to the identity line.

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

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

Dotplot with translocation

Here we translocate a middle portion of the LETTERS array and then make a dot plot of that array with itself. Interestingly, the presence of a translocation does not show up as an abnormality in the dot plot.

seg1 <- LETTERS[1:8]
seg2 <- LETTERS[9:18]
seg3 <- LETTERS[19:26]


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

seqinr::dotPlot(LETTERS.with.transloc,
                LETTERS.with.transloc)

Generating Random Sequences

The sample function allows us to randomly select elements from an array. However, if we do not set the replace parameter to False, there will be repeated elements, as seen below because the selected element will not be kept out of the pool of possible letters for later selections. When we create a dot plot with two random sequences, it shows up as noise and there is no identity line or clear off diagonal lines that indicate shared sequences.

sample(x = LETTERS, size = 26, replace = T)
##  [1] "Q" "X" "S" "Y" "Q" "P" "Z" "Y" "B" "A" "L" "X" "L" "E" "P" "N" "N" "M" "C"
## [20] "Z" "Z" "U" "B" "P" "G" "J"
letters.rand1 <- sample(x = LETTERS, size = 26, replace = F)
letters.rand2 <- sample(x = LETTERS, size = 26, replace = F)


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”

Here, we are fetching two sequences from the NCBI database and then cleaning them.

# 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 = T) # add code
ulcerans_vector <- compbio4all::fasta_cleaner(ulcerans_fasta, parse = T)# add code

Dotplot of sequences

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)

Here, there is a clear identity line running diagonally that indicates great similarity between the two sequences.

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.