Example Mitochondrial cytochrome b – transport electrons From NCBI protein web page, search for cytb and Loxodonta africana (African elephant) Elephas maximus (Indian elephant) Mammuthus primigenius (Siberian wooly Mammoth) Which modern elephant is closer to a mammoth ? Use clustalW to do the alignment Chap. 3: Sequence Alignment
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Example Mitochondrial cytochrome b – transport electrons From NCBI protein web page, search for cytb and Loxodonta africana (African elephant) Elephas.
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Example Mitochondrial cytochrome b –
transport electrons From NCBI protein web page, search
for cytb and Loxodonta africana (African elephant) Elephas maximus (Indian elephant) Mammuthus primigenius (Siberian wooly Mammoth)
Which modern elephant is closer to a mammoth ?
Use clustalW to do the alignment
Chap. 3: Sequence Alignment
>0012AAX12542.1| cytochrome b [Elephas maximus]MTHTRKSHPLFKIINKSFIDLPTPSNISTWWNFGSLLGACLITQILTGLFLAMHYTPDTMTAFSSMSHICRDVNYGWIIRQLHSNGASIFFLCLYTHIGRNIYYGSYLYSETWNTGIMLLLITMATAFMGYVLPWGQMSFWGATVITNLFSAIPYIGTNLVEWIWGGFSVDKATLNRFFAFHFILPFTMVALAGVHLTFLHETGSNNPLGLTSDSDKIPFHPYYTIKDFLGLLILILLLLLLALLSPDMLGDPDNYMPADPLNTPLHIKPEWYFLFAYAILRSVPNKLGGVLALLLSILILGLMPLLHTSKHRSMMLRPLSQVLFWALTMDLLMLTWIGSQPVEYPYIAIGQMASILYFSIILAFLPIAGMIENYLIK
>gi|56578537|gb|AAW01445.1| cytochrome b [Loxodonta africana]MTHIRKSYPLLKIINKSFIDLPTPSNISAWWNFGSLLGACLITQILTGLFLAMHYTPDTMTAFSSMSHICRDVNYGWIIRQLHSNGASIFFLCLYTHIGRNIYYGSYLYSETWNTGIMLLLITMATAFMGYVLPWGQMSFWGATVITNLFSAIPYIGTNLVEWIWGGFSVDKATLNRFFALHFILPFTMTALAGVHLTFLHETGSNNPLGLTSDSDKIPFHPYYTIKDFLGLLILILLLLLLALLSPDMLGDPDNYMPADPLNTPLHIKPEWYFLFAYAILRSVPNKLGGVLALFLSILILGLMPLLHTSKYRSMMLRPLSQVLFWTLTMDLLMLTWIGSQPVEYPYTIIGQMASILYFSIILAFLPIAGMIENYLIK
>gi|2924604|dbj|BAA25008.1| cytochrome b [Mammuthus primigenius]MTHIRKSHPLLKILNKSFIDLPTPSNISTWWNFGSLLGACLITQILTGLFLAMHYTPDTMTAFSSMSHICRDVNYGWIIRQLHSNGASIFFLCLYTHIGRNIYYGSYLYSETWNTGIMLLLITMATAFMGYVLPWGQMSFWGATVITNLFSAIPYIGTDLVEWIWGGFSVDKATLNRFFALHFILPFTMIALAGVHLTFLHETGSNNPLGLTSDSDKIPFHPYYTIKDFLGLLILILFLLLLALLSPDMLGDPDNYMPADPLNTPLHIKPEWYFLFAYAILRSVPNKLGGVLALLLSILILGIMPLLHTSKHRSMMLRPLSQVLFWTLATDLLMLTWIGSQPVEYPYIIIGQMASILYFSIILAFLPIAGMIENYLIK
Pairwise sequence alignment is the most fundamental operation of bioinformatics
It is used to decide if two proteins (or genes) are related structurally or functionally
It is used to identify domains or motifs that are shared among proteins
It is the basis of BLAST searching (next) It is used in the analysis of genomes
Globin Globins carry oxygens and are first proteins to be
sequenced Hemoglobins – in read blood cell Myoglobin – in muscle cells of mammals Leghemoglobin – in legumes (beans, etc.)
Observation or measurement of resemblance, independent of the source of the resemblance
Can be observed now but involves no historical hypothesis
Homology Specifies that sequences and the organisms
descended from a common ancestor Implies that similarities are shared ancestral
characteristics Cannot make the assertion of homology from
historical evidence, and thus is an inference from observations of similarity
Homology Similarity attributed to descent from a common
ancestor Two types of homology
Orthologs Homologous sequences in different species that arose from a
common ancestral gene during speciation; may or may not be responsible for a similar function.
Paralogs Homologous sequences within a single species that arose by
gene duplication.
Orthologs:members of a gene (protein)family in variousorganisms.This tree showsglobin orthologs.
Paralogs: members of a gene (protein) family within aspecies. This tree shows human globin paralogs.
Orthologs and paralogs are often viewed in a single tree
Globin phylogeny by Dayhoff (1972)
Globin phylogeny by Dayhoff in evolutionary time (1972)
Direct Alignment
Given two sequences +1 if letters in the same positions match -1, otherwise
Extremely simple, but what if there is a gap? Gap when a base is inserted or deleted (indel) Maybe only in biological data Maybe more significant mutation – give more
negative score as a penalty
RNDKPFSTARNRNQKPKWWTA+ + - + +- - - - - -
Visual Alignment -- Dotplot
A seq. in x axis and the other in y axis Dot on a crosspoint if
identical in both sequences
view
Special Dotplot
Periodic Palindrome
Sequence Alignment Direct alignment
An alignment with gaps
What is the criteria for a good alignment ? Use score to check for optimality May not produce a unique optimal alignment
g c t g a a c gc t a t a a t c
g c t g - a a - c - g- - c t - a t a a t c
g c t g - a a - c g- c t a t a a t c -
Calculation of an alignment score
General approach to pairwise alignment
Given two sequences Select an algorithm that generates a score Allow gaps (insertions, deletions) Score reflects degree of similarity Alignments can be global or local Estimate probability that the alignment occurred by
chance
Pairwise alignment: protein sequencescan be more informative than DNA
protein is more informative (20 vs 4 characters); many amino acids share related biophysical properties
codons are degenerate: changes in the third position often do not alter the amino acid that is specified
protein sequences offer a longer “look-back” time DNA sequences can be translated into protein, and then
used in pairwise alignments Many times, DNA alignments are appropriate when
to confirm the identity of a cDNA to study noncoding regions of DNA to study DNA polymorphisms example: Neanderthal vs modern human DNA
Genetic Code
Scoring Matrix Dotplot
Incredibly useful in identifying biological significance and interesting regions
Do not privde a measure of statistical similarity A numerical method
Not just provide position-by-position overlap But provide the nature and characteristics of residues
being aligned Scoring matrices
Empirical weighting schemes
Scoring Matrix Three biological factors in constructing a
scoring matrix Conservation
Account for conservation between proteins, but provide a way to assess conservation substitutions
Score represents what residues are capable of substitution for other residues while not adversely affecting the function of the native protein (determined by charge, size, hydrophobicity, etc.)
Frequency Reflect how often residues occur among
proteins Rare residues are given more weight
Evolution By design, implicitly represent evolutionary
Example of scoring matrix Match: +1; mismatch: -μ; indels: -σ
Indels are frequent, and gap penalties proportional to indel sizes are considered to be severe Affine gap penalties soften the penalty rate Can be linear, -(a + bx) for the indel length of x
si-1, j - σsi-1, j = max [ si, j-1 - σ ] si-1, j-1 + 1, if vi=wj
si-1, j-1 - μ, otherwise
Needleman-Wunsch, 1970
Setting up a matrix
Setting up a matrix
Scoring the matrix
Identifying the optimal alignment
Local Alignment
Global sequence alignment is useful for alignment of sequences from the same protein family, for example
Substrings from two sequences may be highly conserved in biological applications Temple Smith and Michael Waterman, 1981 Biologically irrelevant diagonal matches are likely
to have a higher score
Local Alignment Problem Given two strings v and w, and a scoring
matrix δ Find substrings of v and w whose global
alignment is maximal among all substrings of v and w Seemingly harder, because the global alignment
is to find the longest path from (0,0) to (n,m), whereas the local alignment is to find the longest path among all paths between two arbitrary points, (i,j) to (i’, j’)
Add edges of weight 0 from (0,0) to every other vertex (vertex (0,0) is a predecessor of every vertex
Local Alignment Solution
Recurrence kernel becomes
Select the largest si, j
Other non-maximal local alignments may have biological significance Select k best nonoverlapping local alignments