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Sequencing a genome and Basic Sequence Alignment. Lecture 10. Introduction. Annotation of DNA sequences Discovering genomes the shot-gun approach Sequence alignment and sequence matching. Annotation of sequences.

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Presentation Transcript
introduction
Introduction
  • Annotation of DNA sequences
  • Discovering genomes the shot-gun approach
  • Sequence alignment and sequence matching
annotation of sequences
Annotation of sequences
  • As discussed before when the 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

sequence similarity
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.
sequence alignment pair wise a simple global match
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

a simple global match
A simple global Match
  • The non matches are presumed to correspond to mutations; in this case a substitution mutation.
  • In DNA (nucleic acids) mutations
    • Atransition 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)
global sequence alignment different size sequences
Global sequence alignment: different size sequences

A Global alignment between sequence of difference sizes requires the inclusions of gaps [dash] in order to optimise the matching process.

In Example 1(only considers substitution mutations)produces a much lower number of matches than

Example 2 which considers all types of 3 types of point mutations.

This examples calculates a simple matching score;

in DNA you would need to factor in the relative probability of substitutions.

In amino acids the calculation is more complicated.

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)

Global Sequence

example of dna sequence alignment
Example of DNA sequence alignment

Adapted from Klug p. 384

Global Sequence

sequence alignment amino acids
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.

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

sequence alignment a local match
Sequence Alignment: a local Match

A 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.
  • 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

sequence alignment pairwise a motif match
Sequence Alignment: pairwise : a motif match
  • A motif match can find:
  • a “perfect match between a small sequence and 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 “contigiuos ; it can also include conserved distributed pattern
  • You are not from Cork
  • You are not normal
  • They are not happy about…
  • ******

Global Sequence

multiple sequence alignment
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 “canisfamiliaris”
  • 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…. From this determine phylogenetic relationships. ( evolution: refer to understanding bioinformatics chapter 7)

Global Sequence

genomes sequencing and assembling
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

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

Adapted from [1] p. 377

Global Sequence

overlapping fragments example
Overlapping Fragments: example
  • Original sentence:
      • This is the school of computing bioinformatics course.
  • Cut 2 copies of the sentence into fragments
      • This is
      • The school of
      • Computing bioinformatics course
      • This is the
      • School of computing
      • Bioinformatics course

Global Sequence

overlapping fragments example1
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

example of contigs alignment
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

example 2
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,\nAndall
    • their entrances,\nand one man
    • in his time plays many parts.
    • merely players;\nThey have
example 2 solution
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
example 2 solution in detail
Example 2 Solution in detail.
  • the men and womenmerely players;(\n)
  • one man in his time
  • All the world\'s
  • their entrances,(\n) and one man
  • stage, (\n) And allthe men and women
  • They have their exits and their entrances,(\n)
  • world\'sa stage, (\n) And all
  • their entrances, (\n) and one man
  • in his time plays many parts.
  • merely players; (\n) They have
  • 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,
  • And all the men and women merely players;
  • They have
  • 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 all the men and women merely players;
  • They have their exits and their entrances
  • And one man
  • 8: all the world’s a stage,
  • And all the men and women merely players;
  • They have their exits and their entrances
  • And one man
  • 2: all the world’s a stage,
  • And all the men and women merely players;
  • They have their exits and their entrances
  • And one man in his time
  • 9: 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
algorithm to join contigs
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)
potential exam question
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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