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Sequencing a genome and Basic Sequence Alignment

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1 Sequencing a genome and Basic Sequence Alignment
Lecture 11 Global Sequence

2 Introduction Annotation of DNA sequences
Sequence alignment and sequence matching Determining genomes sequences: Supplementary lecture the shot-gun approach

3 Annotation of sequences
As discussed before when a gene sequence’s (DNA and/or mRNA) have been determined (obtained) then the data must be annotated: (Klug 2010) what sequences correspond UTR, exons/ introns, coding sequences (cds), polyA signal Other sequences of interest include: promoters sites and other regulatory regions (enhancers…) Annotation also contains important supplementary material; other organisms that have the same gene; the corresponding protein sequence and journal articles related to the sequences…. Global Sequence

4 Sequence similarity In many cases of the annotation of gene sequence; a sequence homology “test”, to existing sequences whose function is known, is performed. the assumption is that the both sequences were homologous [ have a common ancestor; were the same sequence] but are now different because of a series Mutations: substitution, deletions, insertions The basic concepts behind this process is sequence alignment and determining the strength of the match for the aligned sequence.

5 Sequence Alignment ( Pair-wise) : A simple global match
The assignment of residues-residue corresponds: A Global match: align all of one sequence with another . The figure shows to sequences of nucleic acids. Some have the same base (nucleic acid ) and so there is a match at this position between the strands. This is represented by a vertical line and a blue highlight. Others do not match and have no vertical line and no blue highligh This figure adapted from Klug is a comparison of a “leptin gene” from a dog (top) and a homo sapiens (bottom) Global Sequence

6 A simple global Match The non matches are presumed to correspond to mutations; in this case a substitution mutation. In DNA (nucleic acids) mutations A transition A <-> G is more probable than a transversions T <-> C The substitution mutation is more probable than insertion/deletion. The relative probability of such mutations has to be taken into account when determining the strength of the match. (we will discuss this in greater detail later)

7 Global sequence alignment: different size sequences
Example 1 I am from Cork I am not from Cork **** (4 matches out of 18; based on length of bottom string) A Global alignment between sequence of difference sizes requires the inclusions of gaps [dash] in order to optimise the matching process: the gaps refer to insertion/deletion mutations In Example 1 (only considers substitution mutations) produces a much lower number of matches than ( 4 out of 18) Example 2 which considers all types of the 3 types of point mutations: it aligns the two sequneces to give the maximum matching score. This examples calculates a simple sequencing matching score; in DNA you would need to factor in the relative probability of mutations. In amino acids the calculation is more complicated due to different types of mutations . Example 2 I am ---- from Cork I am not from Cork **** ********** (14 matches out of 18; based on length of bottom string) Global Sequence

8 An Example of DNA sequence alignment
Determine the matching score. Adapted from Klug p. 384 Global Sequence

9 Sequence alignment: Amino Acids
“*” match; “-” gap; “:” conserved substitution “.”semi-conserved substitution. In DNA the sequence “itself” is most important; All nucleic acids have the “same” basics properties. However amino acid sequences produce a 3-D structure, which relates to the property of amino acids in the sequence. So the properties of the amino acids is important Amino Acids with similar, side chain, properties will have overlapping “effects” on 3-D structure of the protein. The above figure takes this into account by referring to two types of substitutions: conserved and semi-conserved substitutions Global Sequence

10 Sequence Alignment: a local Match
A simple local Match example Find a region in one sequence that matches a region in the other. A local match is generally used if there is a larger difference in size between the sequences The overhangs at the beginning and end of the query string are not treated as gaps. Since the local is only being testing for its alignment of a sub-sequence of the global In the example A global (alignment) gives a score of 9 out of 13; A Local (alignment) gives a score is 8 out of 10 ( do not count overhangs…) In general the Alignment with the highest score is the one that is taken. Global Sequence

11 Sequence Alignment: pairwise : a motif match
A motif match can find: a “perfect match between a small sequence of one or more regions in a larger sequence. This plays an important part in looking for repeating sequences [tandem repeats] , and important other “small” sequences; The motif match like the others of course does not have to be “contiguous ; it can also include a conserved distributed pattern. (understanding bioinformatics chapter ) You are not from Cork You are not normal They are not happy about… *** *** Global Sequence

12 Multiple sequence alignment
Similar to the previous except you look for areas conserved between all the sequences in the alignment: My name is denis and I am from cork My name is kieran and I am not from cork We name the dog “canis familiaris” name used to align multiple sequences which can be used to check for conserved motifs/sequences in many species: used to determine protein functionality, promoter signals, enhancer and silencer regions…. Multiple alignment can also determine phylogenetic relationships. (evolution: refer to understanding bioinformatics chapter 7) Global Sequence

13 GENOMES: Sequencing and assembling
The supplementary lecture covers how to produce and determine the sequence of DNA strands. However, the size of the Strands are limited to a few 1000 base pairs. To sequence an organism’ s entire genome : Must use the “shot gun” approach Cut the genome into small fragments whose sequence can be determined. use computational techniques (sequence alignment) to join them back together in the correct order Global Sequence

14 Shot-gun Shot gun approach requires:
Two genetic technologies: (refer to supplementary material for more detail) Restriction enzymes: cut up denatured (ss)DNA Fast DNA sequencing of fragments (sequences) one computational technique (overlapping contigs ) : Combining overlapping contiguous DNA sequences

15 Example of Contigs alignment:
The above diagram shows an DNA example of how overlapping contiguous sequences are aligned. However it is an oversimplification as actual segments are many times larger than shown and overlapping does not always happen at then end of ends of segments. Adapted from: Klug 7th p 378 Global Sequence

16 Overlapping Contiguous Fragments
Adapted from [1] p. 377 Global Sequence

17 Overlapping Fragments: example
Original sentence: This is the school of computing bioinformatics course. Cut “2” copies of the sentence into “random sized” fragments This is The school of Computing bioinformatics course This is the School of computing Bioinformatics course Global Sequence

18 Overlapping Fragments: example
Check for overlaps (prefix and suffix) This is This is the The school of School of computing computing bioinformatics course Bioinformatics course Result of alignment of fragments is: This is the school of computing bioinformatics course Global Sequence

19 Algorithm to join contigs
we need two relationships between fragments:# (1) which fragment shares no prefix with suffix of another fragment# (This tells us which fragment comes first) (2) which fragment shares longest suffix with a prefix of another# (This tells us which fragment follows any fragment)

20 Example 2: Reconstruct the following fragments
the men and women merely players;\none man in his time All the world's their entrances,\nand one man a stage,\nAnd all the men and women They have their exits and their entrances,\n world's a stage,\nAnd all in his time plays many parts. merely players;\nThey have

21 Example 2 Solution all the world’s a stage,
And all the men and women merely players; They have their exits and their entrances And one man plays many parts Order of statements joining together are: 3,7,5,1,10, 6,4,8,2,9

22 Example 2 Solution in detail.
6: all the world’s a stage, And all the men and women merely players; They have their exits and their entrances 4: all the world’s a stage, And one man 8: all the world’s a stage, 2: all the world’s a stage, And one man in his time 9: all the world’s a stage, And one man plays many parts the men and women merely players;(\n) one man in his time All the world's their entrances,(\n) and one man stage, (\n) And all the men and women They have their exits and their entrances,(\n) world's a stage, (\n) And all their entrances, (\n) and one man in his time plays many parts. merely players; (\n) They have Solution Part 1: Order of the statements 3: all the world’s, 7: all the world’s a stage, And all 5: all the world’s a stage, And all the men and women 1: all the world’s a stage, And all the men and women merely players; 10: all the world’s a stage, They have

23 Potential Exam question
Briefly describe the three main types of sequence alignment (6 marks) Explain how would determine the DNA sequence of a genome given that technology can only determine the DNA sequences of relatively small DNA strands (14 marks). Explain, two important elements, of an algorithm that can solve the problem (10 marks)


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