Dotplots for Bioinformatics
- 1. Dot plots Dr Avril Coghlan alc@sanger.ac.uk Note: this talk contains animations which can only be seen by downloading and using ‘View Slide show’ in Powerpoint
- 2. Dot plots • How can we compare the human & Drosophila melanogaster Eyeless protein sequences? One method is a dotplot • A dotplot is a graphical method for assessing similarity Make a matrix (table) with one row for each letter in sequence 1, & one column for each letter in sequence 2 Colour in each cell with an identical letter in the 2 sequences Regions of local similarity between the 2 sequences appear as diagonal lines of coloured cells (‘dots’)
- 3. eg. for sequences ‘RQQEPVRSTC’ and ‘QQESGPVRST’: Q Q E S G P V R S T Sequence 2 R Q Q E Sequence 1 P V R S T C Regions of local similarity between the 2 sequences appear as diagonal lines Some off-diagonal dots may be due to chance similarities
- 4. Problem • Make a dot-plot for DNA sequences “GCATCGGC” & “CCATCGCCATCG”. Are there regions of similarity?
- 5. Answer • Make a dot-plot for DNA sequences “GCATCGGC” & “CCATCGCCATCG”. Are there regions of similarity? C C A T C G C C A T C G G C A T C G G C CATCG in sequence 1 appears twice in sequence 2
- 6. Dot plots with thresholds • If you colour in all cells with an identical letter, some dots may be due to chance similarities • Therefore, it is common to use a threshold to decide whether to plot a ‘dot’ in a cell A window of a certain size (eg. window size = 3) is moved up all possible diagonals, one-by-one A score is calculated for each position of the window on a diagonal : the number of identical letters in the window If the score is equal to or above the threshold (eg. threshold = score of 2), all the cells in the window are coloured in The choice of values for the window size and threshold for the dot plot are chosen by trial-and-error
- 7. eg. for sequences “GCATCGGC” and “CCATCGCCATCG” , using a window size of 3, and a threshold of ≥2: C C A T C G C C A T C G G C A T C G G C Score = 2, ≥ threshold → colour in 3, < 0, 1, = the sliding window and so on....
- 8. Real data: fruitfly & human Eyeless • A dot plot of fruitfly & human Eyeless proteins: Fruitfly Eyeless Window-size = 10, Threshold = 3 Human Eyeless Do you think we chose a good value for the window-size and threshold?
- 9. Real data: fruitfly & human Eyeless • Here is a dot plot of fruitfly and human Eyeless proteins, made using windowsize=10, threshold=5: Fruitfly Eyeless Window-size = 10, Threshold = 5 Human Eyeless Are there any regions of similarity?
- 10. Pros and cons of dot plots • Advantages A dot plot can be used to identify long regions of strong similarity between two sequences It produces a plot, which is easy to make and to interpret It can be used to compare very short or long sequences (even whole chromosomes – millions of bases) • Disadvantages It is necessary to find the best window size and threshold by trial-and- error A dot plot can only be used to compare 2 sequences, not >2 sequences It doesn’t tell you what mutations occurred in the region of similarity (if there is one) since the two sequences shared a common ancestor
- 11. Software for making dotplots • dotPlot() function in the SeqinR R library Allows you to specify a windowsize and threshold If the score in a window is ≥ than the threshold, colours in the 1st cell in the window (not all cells) • EMBOSS dottup Allows you to specify a windowsize but not a threshold If all cells in a window are identities, it colours in all cells in the window • EMBOSS dotmatcher Allows you to specify a windowsize and threshold Instead of using the number of identities in a window as the window score, it calculates a more complex score based on the similarities of the bases/amino acids
- 12. Problem • Make a dot-plot for amino acid sequences “RQQEPVRSTC” and “QQESGPVRST”, using a window size of 3, and a threshold of ≥3
- 13. Answer • Make a dot-plot for sequences “RQQEPVRSTC” and “QQESGPVRST”, using window size: 3, threshold: ≥3 Q Q E S G P V R S T R Q Q E P V R S T C
- 14. Further reading • Chapter 3 in Introduction to Computational Genomics Cristianini & Hahn • Practical on dotplots in R in the Little Book of R for Bioinformatics: https://a-little-book-of-r-for- bioinformatics.readthedocs.org/en/latest/src/chapter4.html
Editor's Notes
- #4: In R: setwd("C:/Documents and Settings/Avril Coughlan/My Documents/BACKEDUP/MScCourseLectures/MB6301Lectures/MB6301_Ls3456_Aln") library("seqinr") seq1 <- “RQQEPVRSTC” seq2 <- “QQESGPVRST” seq1b <- s2c(seq1) seq2b <- s2c(seq2) source(“dotplot.R”) makeDotPlot1(seq1b,seq2b,dotsize=1)
- #6: In R: setwd("C:/Documents and Settings/Avril Coughlan/My Documents/BACKEDUP/MScCourseLectures/MB6301Lectures/MB6301_Ls3456_Aln") library("seqinr") seq1 <- “GCATCGGC” seq2 <- “CCATCGCCATCG” seq1b <- s2c(seq1) seq2b <- s2c(seq2) source(“dotplot.R”) makeDotPlot1(seq1b,seq2b,dotsize=1)
- #8: In R: setwd("C:/Documents and Settings/Avril Coughlan/My Documents/BACKEDUP/MScCourseLectures/MB6301Lectures/MB6301_Ls3456_Aln") library("seqinr") seq1 <- “GCATCGGC” seq2 <- “CCATCGCCATCG” seq1b <- s2c(seq1) seq2b <- s2c(seq2) source(“dotplot.R”) makeDotPlot2(seq1b,seq2b,dotsize=1,windowsize=3,threshold=2)
- #9: setwd("C:/Documents and Settings/Avril Coughlan/My Documents/BACKEDUP/MScCourseLectures/MB6301Lectures/MB6301_Ls3456_Aln") library("seqinr") seq1 <- read.fasta(“human.fa”) # human Eyeless seq2 <- read.fasta(“fly.fa”) # fruitfly Eyeless seq1b <- seq1[[1]] seq2b <- seq2[[1]] source(“dotplot.R”) makeDotPlot2(seq1b,seq2b,dotsize=1,windowsize=10,threshold=3) Saved picture as dotplot2.png
- #10: setwd("C:/Documents and Settings/Avril Coughlan/My Documents/BACKEDUP/MScCourseLectures/MB6301Lectures/MB6301_Ls3456_Aln") library("seqinr") seq1 <- read.fasta(“human.fa”) # human Eyeless seq2 <- read.fasta(“fly.fa”) # fruitfly Eyeless seq1b <- seq1[[1]] seq2b <- seq2[[1]] source(“dotplot.R”) makeDotPlot2(seq1b,seq2b,dotsize=1,windowsize=10,threshold=5) Saved picture as dotplot1.png
- #14: In R: setwd("C:/Documents and Settings/Avril Coughlan/My Documents/BACKEDUP/MScCourseLectures/MB6301Lectures/MB6301_Ls3456_Aln") library("seqinr") seq1 <- "RQQEPVRSTC" seq2 <- "QQESGPVRST" seq1b <- s2c(seq1) seq2b <- s2c(seq2) source("dotplot.R") makeDotPlot2(seq1b,seq2b,dotsize=1,windowsize=3,threshold=3)