Sequence Alignment Fundamentals
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Which of the following is the primary reason for performing sequence alignment?

  • Functional prediction based on identifying homologous proteins or protein domains. (correct)
  • To determine the evolutionary distance between species.
  • To calculate the length of the sequences.
  • To identify exact matches between sequences.

A sequence similarity of less than 20% almost always indicates functional dissimilarity.

False (B)

Name one common algorithm used for sequence alignment.

BLAST

In sequence alignment, a sequence similarity greater than or equal to _____% almost always indicates similarity in function.

<p>30</p> Signup and view all the answers

Which of the following alignment algorithms is mentioned for sequence alignment?

<p>All of the above (D)</p> Signup and view all the answers

In sequence alignment, what is the primary purpose of the scoring matrix?

<p>To assign scores to matches, mismatches, and gaps. (B)</p> Signup and view all the answers

In the provided alignment example, a gap is always penalized with a score of -1, regardless of its position.

<p>True (A)</p> Signup and view all the answers

In the context of sequence alignment, what does 'trace back' refer to?

<p>Identifying the path of optimal alignment decisions.</p> Signup and view all the answers

When constructing the alignment matrix, if the diagonal score (match/mismatch) is higher than the scores from the upper or left cells (gap), you should record the ______ score and include an arrow from the diagonal cell.

<p>diagonal</p> Signup and view all the answers

What is the score at position (1,1) of the matrix, representing the alignment of 'G' in sequence 1 with 'G' in sequence 2?

<p>1 (B)</p> Signup and view all the answers

According to the provided rules, it is possible to have multiple arrows pointing to the same cell in the alignment matrix.

<p>False (B)</p> Signup and view all the answers

Within dynamic programming for sequence alignment, explain how the choice of scoring parameters (match, mismatch, gap penalties) can affect the resulting alignment.

<p>The choice influences the number of gaps and mismatches in the optimal alignment.</p> Signup and view all the answers

Match the following alignment steps with their descriptions:

<p>Scoring Matrix = Defines the scores for matches, mismatches, and gaps. Trace Back = Determines the optimal alignment path from the matrix. Alignment = The final result of the alignment process. Gap Penalty = Subtraction for the addition of a gap.</p> Signup and view all the answers

In the context of sequence alignment using a scoring matrix, what is the primary purpose of setting the first row and first column to zero?

<p>To allow local alignments to start at any position within the sequences. (B)</p> Signup and view all the answers

In a local sequence alignment scoring matrix, negative values are allowed at intermediate steps to represent mismatches and gaps, and these negative values are retained for trace back.

<p>False (B)</p> Signup and view all the answers

In sequence alignment, what is the significance of the 'trace back' step, and where does it terminate in a local alignment?

<p>The trace back step identifies the optimal alignment path by following the highest scores in the matrix, and it terminates at a cell with a score of zero in local alignment.</p> Signup and view all the answers

In constructing a local alignment scoring matrix, the value of a cell representing a match/mismatch is calculated based on the diagonal cell, with a score added for a match or subtracted for a ________.

<p>mismatch</p> Signup and view all the answers

Which type of sequence alignment is most suitable for identifying regions of similarity between sequences with significant variations and differing lengths?

<p>Local alignment (C)</p> Signup and view all the answers

Match the actions with which cell they affect in the matrix.

<p>Box beside (+Gap) = Left cell Box upper (+Gap) = Top cell Diagonal (+Match/Mismatch) = Diagonal cell</p> Signup and view all the answers

In sequence alignment, a positive score always indicates a desirable alignment, such as a match, while a negative score invariably indicates an undesirable alignment, such as a mismatch or gap.

<p>False (B)</p> Signup and view all the answers

In dynamic programming for sequence alignment, what is the purpose of the 'trace back' step?

<p>To determine the optimal alignment path</p> Signup and view all the answers

In sequence alignment scoring, a ______ is typically assigned a negative score to penalize the introduction of spaces in the alignment.

<p>gap</p> Signup and view all the answers

Using a scoring system where a match = +1, mismatch = -1 and gap = -1, which of the following alignments of ATGC to TGC yields the highest score?

<p>ATGC _TGC (A)</p> Signup and view all the answers

Match the components of global sequence alignment with their descriptions:

<p>Scoring matrix = Defines scores for matches, mismatches, and gaps. Trace back = Identifies the optimal alignment path through the matrix. Alignment = The final result showing the correspondence between sequences.</p> Signup and view all the answers

What is the primary purpose of a dot plot in sequence comparison?

<p>To visually identify regions of similarity between two sequences (A)</p> Signup and view all the answers

Which statement best describes the significance of regions of local similarity located on the diagonal in a dot plot?

<p>They indicate conserved regions or domains between the sequences. (A)</p> Signup and view all the answers

Which of the following steps is crucial for conducting a BLAST search after selecting the appropriate program?

<p>All of the above. (D)</p> Signup and view all the answers

In a BLAST output, a '+' symbol between amino acid residues indicates an exact amino acid match between sequences.

<p>False (B)</p> Signup and view all the answers

Define the purpose of the E-value in the context of BLAST results.

<p>The E-value represents the probability that a match as good as, or better than, the one found would occur by chance alone. Lower E-values indicate more significant matches.</p> Signup and view all the answers

When analyzing a protein sequence using BLAST, the initial step involves identifying the appropriate BLAST ______ to use.

<p>program</p> Signup and view all the answers

In the context of sequence alignment, what does a 'gap' generally represent?

<p>An insertion or deletion in one sequence relative to another. (A)</p> Signup and view all the answers

Match each term related to BLAST with its corresponding definition or function:

<p>Query Sequence = The sequence submitted for comparison against databases. Database = A collection of known sequences used for comparison. E-value = A statistical measure of the significance of a match. BLAST = A suite of algorithms for comparing biological sequences.</p> Signup and view all the answers

In a sequence alignment scoring matrix, a negative score always indicates an error in the alignment.

<p>False (B)</p> Signup and view all the answers

What is the purpose of the 'trace back' step in a local sequence alignment algorithm?

<p>To identify the path of highest score which represents the optimal local alignment</p> Signup and view all the answers

In a scoring matrix used for sequence alignment, a higher score generally indicates a ______ match between the sequences.

<p>better</p> Signup and view all the answers

Match the alignment term with its definition:

<p>Gap Penalty = A score deducted for introducing a gap in the alignment. Mismatch = Occurs when two bases at the same position in an alignment are different. Local Alignment = Finds the best matching region(s) between two sequences. Scoring Matrix = A table of values that define the cost of matches, mismatches, and gaps.</p> Signup and view all the answers

If you have a scoring matrix where a match = +2, mismatch = -1, and gap = -2, what is the score for aligning AT with A- (where - denotes a gap)?

<p>0 (D)</p> Signup and view all the answers

Global and local alignment algorithms always produce the same alignment for any given pair of sequences.

<p>False (B)</p> Signup and view all the answers

In the provided example of local alignment, which rule is essential for finding the optimal alignment path in the scoring matrix?

<p>Tracing back from the highest score to a zero value. (B)</p> Signup and view all the answers

Flashcards

Sequence Alignment

Arranging sequences to highlight regions of similarity.

Why Align Sequences?

To find sequence similarity to predict protein functions.

Sequence Similarity = Function Similarity

Sequence similarity often indicates similar function and/or 3D structure.

Alignment Algorithms

Algorithms like Dot Matrix, Dynamic Programming, BLAST, FASTA, and Clustal.

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Sequence Identity

Percentage of identical residues in aligned sequences.

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Dot Plot

A graphical method to visually assess the similarity between two sequences. Local similar regions appear as diagonal lines or clusters.

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Global Alignment

An alignment that finds the best match across the entire length of two sequences.

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Local Alignment

An alignment that identifies regions of similarity within sequences, disregarding overall sequence homology.

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Dynamic Programming

A computational technique used to find the optimal alignment between sequences by considering all possible alignments and assigning scores.

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Scoring Matrix

A matrix that assigns values to matches, mismatches, and gaps during sequence alignment.

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Trace Back

Process of determining the series of edits (matches, mismatches, gaps) needed to transform one sequence into another based on the scoring system.

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Alignment

The final result of sequence alignment, showing the correspondence between residues in the aligned sequences.

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Scoring Alignments

Assign scores based of the parameters (Match, Mismatch and gap), and then compare results to find the best alignment.

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Local vs. Global Alignment

Local alignment finds the best matching segments, while global alignment aligns the entire sequence length.

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Gap Penalty

Represents a space or insertion in a sequence to maximize alignment with another sequence. Has a cost (negative score).

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Gap in Sequence Alignment

Adding a gap to a sequence affects the alignment score.

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Sequence Similarity

The best matches are found by maximizing the alignment score.

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What is BLAST?

A tool to find similar sequences in databases.

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BLAST steps

  1. Find program, 2. Enter query sequence, 3. Select databases, 4. Run BLAST, 5. Analyze output, 6. Interpret E-values.
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What is a Query sequence?

The sequence you submit to BLAST for comparison.

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What is an E-value?

A measure of the likelihood that the match occurred by chance. Lower E-value is better.

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BLAST output symbols

Identical amino acids or nucleotides are shown as letters; similar ones as plus signs (+).

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Diagonal Rule

When performing trace back during sequence alignment, take the value from the diagonal cell (representing a match or mismatch).

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Box Beside (+ Gap)

When performing trace back, consider the score of the cell to the left (introducing a gap in the vertical sequence).

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Local Alignment Scoring

Match=+1, Mismatch=-1, Gap = -1. Used to score sequence alignments.

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Local Alignment Steps

  1. Gap before the first letter. Set the first row and column to zero. 2. Fill the matrix using scoring rules. 3. Trace back from the highest score to zero.
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Scoring Matrix Rules (Local)

From each cell, find the maximum score using these conditions: 1. From the left cell + gap penalty, 2. From the top cell + gap penalty 3. From the diagonal cell + (match or mismatch score), 4. Zero. Keep only the positive values.

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Trace Back in Matrix

When backtracking, start from the highest score in the matrix. Follow the path that leads to the highest score until you reach a cell with a score of zero.

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Alignment Construction

Insert gaps to maximize the alignment score. Trace back, matching or mismatching letters to create the optimal alignment, including spaces (gaps).

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Study Notes

  • BT 305 Lecture 5 and 6 covers Sequence Alignment and Basic Alignment Tools.

Sequences

  • Nucleic acids and proteins are sequences that can be aligned.
  • Nucleic acid example sequence: ATGCGCTA.....
  • Protein example sequence: RHKSPK......

Sequence Alignment

  • Involves matching, mismatching, and gaps.
  • A match signifies identical elements at the same position in aligned sequences.
  • A mismatch indicates differing elements at the same position in aligned sequences.
  • A gap represents an insertion or deletion in one sequence relative to another to optimize alignment.
  • Sequence alignment aims to find the best possible match between sequences.
  • The goal is to find the sequence similarity because sequence similarity can indicate functional similarity.

Why Sequence Similarity?

  • Functional prediction is based on the identification of homologous proteins or protein domains.
  • A core assumption is that sequence similarity implies similarity in function and/or 3D structure.
  • A similarity greater than or equal to 30% is almost always functionally relevant.
  • A 20-30% similarity falls into the twilight zone

Alignment Algorithms

  • Dot Matrix
  • Dynamic programming
  • Blast
  • Fasta
  • Clustal

Similarity Strength

  • Similarity strength can be measured by percent identity, percent similarity, and an E-value which is a statistical measure.

Sequence Identity vs Sequence Similarity

  • Sequence identity is the number of residues that are identical in both aligned sequences.
  • Sequence similarity, especially in proteins, means chemically similar residues are interchangeable but not identical.

Dot Plot

  • Dot plots are a graphical method to assess sequence similarity.
  • A dot-plot gives a visual assessment of similarity based on identity.
  • Regions of local similarity appear in the diagonal.

Global vs. Local Alignment

  • Global alignment is best for sequences that are generally similar along their entire lengths.
  • Local alignment is best for finding regions of similarity within sequences that may have dissimilar regions elsewhere.

Dynamic Programming

  • Global alignment covers the entire lengths of the sequences involved
  • The Needleman-Wunsch algorithm finds the best global alignment between two sequences.
  • Local alignment only covers parts of the sequences
  • The Smith-Waterman algorithm finds the best local alignment between 2 sequences.

Scoring Strategy

  • For sequence alignment, a scoring system is used:
  • Align and assign a score for a match which is +1
  • Mismatch is scored as -1
  • A gap is scored as -1

Global Alignment Strategy

  • Sequence 1= GCATGCU and Sequence 2= GATTACA
  • Use a scoring matrix where (match=+1, Mismatch=-1, Gap = -1).
  • Trace back from the highest score to determine the alignment path
  • Align the sequences based on trace back; corner arrow entries equal letters, and the arrow pointing to the sequence equals a gap.
  • Rules:
    • Put a gap before the first letter.
    • Box beside (+Gap)
    • Box upper (+Gap)
    • Diagonal (+Match/Mismatch)
    • Keep the highest and put an arrow from where it came from.

Local Alignment Strategy

  • Sequence 1 = TCG and Sequence 2= ATCG
  • Rules
    • Put a gap before the first letter.
    • Put zero for 1st row and column.
    • Box beside (+Gap)
    • Box upper (+Gap)
    • Diagonal (+Match/Mismatch)
    • Keep only the +ve value. Any (-ve) = 0
    • Trace back stop at zero, ie, Highest => 0

Constructing a Scoring Matrix

  • The construction of scoring matrices relies on statistics and chemical knowledge.

E-Value (Expectation Value)

  • The quality of the alignment is represented by the Score (s).
  • The significance of the alignment is computed as an E- value.
  • E-value measures the number of alignments with scores equivalent to or better than a score s that can be expected to arise by chance in a database of the same size not containing a homologous sequence.
  • A smaller E-value signifies a more significant score.

BLAST

  • BLAST (Basic Local Alignment Search Tool) encompasses various implementations and enhancements to find "High Scoring Pairs" in sequence alignments in databases.
  • FAST
  • Heuristic, not exact, not optimal.
  • It is not very sensitive
  • Is the most common tool that is used in bioinformatics.

BLAST Protocols

  • The most common BLAST search includes five protocols
Program Database Query
BLASTN Nucleotide Nucleotide
BLASTP Protein Protein
BLASTX Protein Nt -> Protein
TBLASTN Nt -> Protein Protein
TBLASTX Nt -> Protein Nt -> Protein

Steps to submit sequences to Blast:

  • Find the appropriate blast programme.
  • Enter the Query sequence.
  • Select the data bases.
  • Run BLAST search.
  • Analyze output.
  • Interpret E-values.

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Explore the core principles of sequence alignment, including its primary purpose and common algorithms. Learn about sequence similarity thresholds and the role of scoring matrices. Understand gap penalties and the trace back process.

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