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Assessment of sequence alignment

Lecture 10

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Introduction

• The Dot plot Matrix visualisation matching tool:– Basics of Dot plot

– Examples of Dot plot matching sequences

– Tandems repeats self matching

– Inverted repeats: genetic palindromes

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Sequence alignment Analysis• In order to measure the degree of similarity

between sequences they must first be aligned to maximise the matching score (refer to lecture 11):

• Example 2• I am ---- from Cork• I am not from Cork• **** **********

• (14 matches out of 18; based on length of bottom string)

• Example 1• I am from Cork• I am not from Cork• **** • (4 matches out of 18; based on

length of bottom string)

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The Dot plot

• A “better” way of doing this is to represent each sequence as a table or matrix, where one sequence represents the rows and the other the columns. The Dot plot Matrix is a visual way of seeing the alignment between two sequences:– The first sequence (query sequence) represents the rows and

the other sequence (subject sequence) represents the columns.

– All elements (row/column) are checked for a match and if there the cell is marked.

– This will show all areas of both sequences where matches occur.

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Dot plot• Consider the following:

– Diagonal lines represent a alignments (match)

– Horizontal lines between aligned sequences indicate gaps are required (where the gaps indicate a deletion/insertion)

–

• This has four “potential” aligned sequences: – D->Y;– H->N– R->0– 0->H

• Longest sequence of alignments are:– “THIS” ; and “SEQUENCE“; – “IS” would be considered as gaps

• The pink dots: they can represent noise (spurious alignments)

adapted from understanding bioinformatics p. 77

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Dot plot Matrix: purpose• This allows us to visualise areas of “local

alignment” as opposed to global alignment. • One of the main purpose to find domains /

motifs that match . This could be useful for many reasons; e.g. promoter factor binding site, finding exons….

• For visualisation of pair-wise alignment you have one query on the x-axis and the other on the y-axis.

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Dot Plot noise

This shows the effect of noise (blue line has be been inserted to highlight alignment if interest. The figure on the left represents SH2 sequence (sample files ) plotted against inself. The one on the right has been filter; in this case an alignment must be at least 10 residues long with a score of 3. adapted from understanding bioinformatics p. 77

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Dot plot Matrix: imperfect match

• Some alignments require gaps to increase the matching score; the gaps are used represent inclusion/deletion mutations

• The diagram shows that most of the 2 sequences are aligned. Where there are gaps indicates areas of non-alignment or mismatches: gaps or substitutions Adapted from: dotplot example

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Dot plot: example 1Refer to saved web page

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Dot plot: example 1

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Dot plot for Tandem Repeats

• The human genome has many tandem repeats small sequences of nucleic acids (bases)/ Amino acids that are repeated and are ubiquitous in genomes and can compromise 50% of genome. (Richard 2008)

• They can be used as genealogical markers• To determine specific regions of interest; e.g. introns• Play a significant part in evolution Gemayel 2010 • An example of a protein with multiple repeats is

human mucin (Baxevanis 2005 p. 297)

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Dot plot of tandem repeats

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Tandem repeat as a sequence

Tandem repeat 1

A B R A C A D A B R A C A D A B R A

A B R A C A D A B R A C A D A B R A

Tandem repeat 2

A B R A C A D A B R A C A D A B R A

A B R A C A D A B R A C A D A B R A

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Tandem repeat dot plot

• To determine if there is tandem repeats the sequence is compared with itself (refer table 1)

• The more diagonals the more repeats• The diagonals at the bottom left compare the

start with the finish• The fact the main diagonal means the both

sequences are the same . • The lines are symmetrical around the main

diagonal:

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Tandem repeats (Example)• BRCA2 gene has a number of BRC repeats (39 residues long. The diagram shows

two plots: one with noise (unfiltered) and the other showing two repeating sequences. Adapted from Figure 4.3 understanding bioinformatics

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Genetic “Palindromes”• A palindrome is a word that is spelt the same from right to left as well as from left to

write: This will give an “X” shaped dot-plot. (try; eye, navan; never odd or even …..)

• Remember left to right is (5’ to 3’) on primary strand and right to left is (5’ to 3’) on the complimentary strand. Alternatively it means a match between a strand and its reverse compliment.

• 2 possible types of “Genetic Palindromes” [the difference being that the left to right, read, is on one strand while the right to left, read, is on its complimentary strand]: – Restrictive enzymes such as EcoR1:

• 5’ GAATTC 3’• 3’ CTTAAG 5’

– Inverted repeats • On different segments; each repeat read the same (GTGAG) but in opposite directions. An example is

promoter region for the CAP protein in the lac operon : – 5‘ GTGAGnnnCTCAC 3'

3' CACTCnnnGAGTG 5’

• What will the dot plot for the above 2 sequences look like.

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Supplementary reading

• The following provides links to further reading on DOT PLOTS. – introduction to dotplot (figure 6 gives a more

indepth view of different types of plots referred to above: alignment, alignment with gaps, tandem repeats, palindromes…..

– Inverted repeats and dotplot. (more advanced analysis of plots for inverted repeats)

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Exam Question• Describe, using a suitable example, how to

construct a dot plot matrix for the alignment of DNA/AA sequences. (10 marks)

• Describe the significance of two types of repeating sequences found in DNA sequences (6 marks)

• Explain, using suitable examples, how the DOT plot matrix can find the two types of repeating regions [what is plotted against what and what will the DOT PLOT look like] (14 marks)

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References

• Baxevanis A.D. 2005 Bioinformatics: a practical guide to the analysis of genes and proteins chapter 11; Wiley

• Klug, W. S. (2010); the essentials of genetics; 7th ed Pearson Education

• Gemayel, R. et al 2010 Variable tandem repeats accelerate evolution of coding and regulatory sequences. Annu Rev genet 44: 445-477

• Richard, G.F. (2008) Comparative genomics and molecular dynamics of DNA repeats in eukaryotes. Microbiol Mol biol rev 2008 Dec;72(4):686-727