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ALIGNMENT OF NUCLEOTIDEALIGNMENT OF NUCLEOTIDE&&

AMINO-ACID SEQUENCESAMINO-ACID SEQUENCES

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An alignment is an evolutionarily meaningful comparison of two or more sequences (DNA, RNA, or proteins).

In the case of two DNA sequences, an alignment consists of a series of paired bases, one base from each sequence. There are three types of pairs:

(1) matches = the same nucleotide appears in both sequences. (2) mismatches = different nucleotides are found in the two sequences. (3) gaps = a base in one sequence and a null base in the other. GCGGCCCATCAGGTAGTTGGTG-G

GCGTTCCATC--CTGGTTGGTGTG***..***** .*.******* *

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Alignment:Alignment: A hypothesis concerning positional homology among residues in a sequence.

Positional homologyPositional homology = A pair of nucleotides from two aligned sequences that have descended from one nucleotide in the ancestor of the two sequences.

GCGGCCCATCAGGTAGTTGGTG-GGCGTTCCATC--CTGGTTGGTGTG***..***** .*.******* *

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Positional homologyPositional homology = A pair of nucleotides from two aligned sequences that have descended from one nucleotide in the ancestor of the two sequences.

GCGGCCCATCAGGTAGTTGGTG-GGCGTTCCATC--CTGGTTGGTGTG***..***** .*.******* *

These two nucleotides are derived from the ancestor of cats and armadillos.

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Homology:Homology: The term was coined by Richard Owen in 1843.

Definition: Similarity resulting from common ancestry.

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Homology: A qualitative statment

• Homology designates a relationship of common descent between entities

• Two genes are either homologs or not– it doesn’t make sense to say “two genes are 43% homologous.”

– it doesn’t make sense to say “Linda is 43% pregnant.”

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By comparing homologous characters, we can reconstruct the evolutionary events that have led to the formation of the extant sequences from the common ancestor.

Homology

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When dealing with sequences, we are interested in POSITIONAL HOMOLOGY.

We identify positional homology by ALIGNMENT.

Homology

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ACTGGGCCCAAATC

1 deletion 1 substitution

1 insertion 1 substitution

ACAGGGCCACAAATCACTGGCCCAGATC

ACTGGCCCAGATC--ACAGGGCCACAAATC**.**.***.*..--

ACT-GGCC-CAGATCACAGGGCCACAAATC**.-****-**.***

Correct alignmentCorrect alignment Incorrect alignmentIncorrect alignment

ACTGGGCCCAAATCG

ACTGGGCCCAAATCA

A

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unknown

ACAGGGCCACAAATCACTGGCCCAGATC

ACTGGCCCAGATC--ACAGGGCCACAAATC**.**.***.*..--

ACT-GGCC-CAGATCACAGGGCCACAAATC**.-****-**.***

Correct alignment?Correct alignment? Incorrect alignment?Incorrect alignment?

unknownunknown

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Sequence alignment = Sequence alignment = The identification of the location of deletion or insertions that might have occurred in either of the two lineages since their divergence from a common ancestor. InInsertionsertion ++ DelDeletionetion == IndelIndel oror GapGap

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Sequence alignment Sequence alignment

1. Pairwise 1. Pairwise alignment alignment

2. Multiple 2. Multiple alignmentalignment

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- Two DNA sequences: A and B.- Two DNA sequences: A and B.- Lengths are - Lengths are mm and and nn, , respectively. respectively. - The number of matched pairs is - The number of matched pairs is xx. . - The number of mismatched pairs - The number of mismatched pairs is is yy. . - Total number of bases in gaps - Total number of bases in gaps is is zz..

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An gap indicates that a deletiondeletion or an insertioninsertion has occurred in one of the two lineages.

GCGG-CCATCAGGTAGTTGGTG--GCGTTCCATC--CTGGTTGGTGTG

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The alignment is the first step in many evolutionary and functional studies.

Errors in alignment tend to amplify in later computational stages.

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Methods of alignment:

1. Manual2. Dot matrix3. Algorithmic (scoring matrices and gap penalties)

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Manual aliManual aliggnmentnment. When there are few gaps and the two sequences are not too different from each other, a reasonable alignment can be obtained by visual inspection.

GCG-TCCATCAGGTAGTTGGTGTGGCGTTCCATCAGGTGGTTGGTGTG*** **********.*********

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Advantages of manual alignment: (1) use of a powerful and trainable tool (the brain, well…, some brains).(2) ability to integrate additional data, e.g., domain structure, biological function (e.g., 3D structure).

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Disadvantages of manual alignment: 1. Subjectivity = the inability to formally specify the algorithm.2. Irreproducibility = the inability of two researchers to reach the same result. 3. Unscalability = the inability to apply the method to long sequences. 4. Incommensurability = the inability to compare the results to those derived from other methods.

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The dot-matrix method: The two sequences are written out as column and row headings of a two-dimensional matrix. A dot is put in the dot-matrix plot at a position where the nucleotides in the two sequences are identical.

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The alignment is defined by a path from the upper-left element to the lower-right element.

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There are 4 possible steps in the There are 4 possible steps in the path: path:

(1) a diagonal step through a dot = match.

(2) a diagonal step through an empty element of the matrix = mismatch.

(3) a horizontal step = a gap in the sequence on the top of the matrix.

(4) a vertical step = a gap in the sequence on the left of the matrix.

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alloweddirections

forbiddendirections

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A dot matrix may become cluttered. With DNA sequences, ~25% of the elements will be occupied by dots by chance alone.

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The number of spurious matches is determined by: window size, stringency, & alphabet size.

window size =1stringency = 1alphabet size = 4

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window size =1stringency = 1alphabet size = 4

window size = 3stringency = 2alphabet size = 4

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window size = 1stringency = 1alphabet size = 20

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Dot-matrix methods:Dot-matrix methods:Advantages: May unravel information on the evolution of sequences.

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Advantages:Highlighting Information

The vertical gap The vertical gap indicates that a indicates that a coding region coding region corresponding to corresponding to ~75 amino acids ~75 amino acids has either been has either been deleted from the deleted from the human gene or human gene or inserted into the inserted into the bacterial gene. bacterial gene.

Window size = 60 amino acids; Stringency = 24 matches

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The two The two diagonally diagonally oriented parallel oriented parallel lines most lines most probably indicate probably indicate that a small that a small internal internal duplication has duplication has occurred in the occurred in the bacterial gene. bacterial gene.

Window size = 60 amino acids; Stringency = 24 matches

Advantages:Highlighting Information

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Dot-matrix Dot-matrix methods:methods:Disadvantage: May not identify the best alignment.

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Scoring Matrices & Gap Penalties

The true alignment between two sequences is the one that reflects accurately the evolutionary relationships between the sequences.

Since the true alignment is unknown, in practice we look for the optimal alignment, which is the one in which the numbers of mismatches and gaps are minimized according to certain criteria.

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Unfortunately, reducing the number of mismatches results in an increase in the number of gaps, and vice versa.

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= matches = mismatches = nucleotides in gaps = gaps

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The scoring scheme comprises a gap penalty and a scoring matrix, M(a,b), that specifies the score for each type of match (a = b) or mismatch (a b).

The units in a scoring matrix may be the nucleotides in the DNA or RNA sequences, the codons in protein-coding regions, or the amino acids in protein sequences.

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If you want to know the secrets behind the black box of sequence alignment, you will have to take a class in BIOINFORMATICS.

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Multiple Sequence

Alignment is infinitely more complicated than

pairwise alignment

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Multiple Sequence

Alignment does not have an

exact optimal solution. It is solved heuristically.

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A Multiple Sequence Alignment

GCGGCTCA TCAGGTAGTT GGTG-G SpinachGCGGCCCA TCAGGTAGTT GGTG-G RiceGCGTTCCA TC--CT-GTT GGTGTG MosquitoGCGTCCCA TCAGCTAGTT GTTG-G MonkeyGCGGCGCA TTAGCTAGTT GGTG-A Human***...** *.--.*-*** *.**-.