1. Assessment of sequence alignment Lecture 10 1

2. Introduction The Dot plot Matrix visualisation matching tool: – Basics of Dot plot – Examples of Dot plot matching 2 sequences – Tandems repeats self matching – Inverted repeats: genetic palindromes 2

3. Sequence alignment Analysis In order to measure the degree of similarity between sequences they must first be aligned to maximise the matching score: 3 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)

4. 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. 4

5. Dot plot Consider the following: – Diagnol 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: – D->Y; and H->Y and Do you think, assuming this represented DNA/AA sequences that “gaps” should be used to join these sequences? 5 adapted from Lesk 2008.

6. Dot plot Matrix 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…. Can you think of any others (refer to previous lecture’s)? 6

7. Dot plot: the previous examples Klug 7ed p. 403; There is sample DNA sequences which we will match via: the blast program on the NCBI website:blast program Go to the exploring genomics part of the books website and cut and paste the sequences into the query and subject “windows” You must ensure that you set the search low. [discussed in next lecture] Run the blast program [ a tool that aligns and measures the alignment (discussed in next lecture)] 7

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9. Dot plot: example 1 9 Refer to saved web page

10. Dot plot: example 1 10

11. Dot plot: Example 2 11

12. Sequence Matching: Example 2 12

13. 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)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 2010Gemayel 2010 An example of a protein with multiple repeats is human mucin (Baxevanis 2005 p. 297) 13

14. Dot plot of tandem repeats 14

15. Tandem repeat as a sequence Tandem repeat 1 ABRACADABRACADABRA ABRACADABRACADABRA Tandem repeat 2 ABRACADABRACADABRA ABRACADABRACADABRA 15

16. 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: 16

17. 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. ( 17

18. Using dot plot and BLAST Refer to fig 9.19 understanding bioinformatics which shows how the DOT plot and a BLAST can be used to find longer alignments. 18

19. Exam Question Describe how to construct a dot plot matrix for the alignment of DNA/AA sequences and explain how they can be used to check for the presence of different types repeating sequences. 19

20. 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; 7 th 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 More general DOT PLOT information: introduction to dotplotintroduction to dotplot Inverted repeats and dotplot. Inverted repeats and dotplot 20