Sequence alignment fundamentals MCQs With Answer

Introduction: This quiz collection on Sequence Alignment Fundamentals is tailored for M.Pharm students to reinforce essential bioinformatics concepts applied in drug discovery and molecular analysis. It covers pairwise and multiple sequence alignment principles, dynamic programming algorithms (Needleman–Wunsch and Smith–Waterman), scoring systems including substitution matrices (PAM/BLOSUM), gap penalties, and practical tools such as BLAST, PSI-BLAST, and profile HMMs. Each question emphasizes conceptual understanding and practical interpretation so you can evaluate homology, functional conservation, and evolutionary relationships relevant to pharmacology, target identification, and comparative genomics. Use these MCQs to test and strengthen your analytical skills in sequence comparison and interpretation.

Q1. What is the primary purpose of sequence alignment in bioinformatics?

• To arrange sequences randomly for data storage
• To determine the three-dimensional structure directly from sequence
• To identify regions of similarity that may indicate functional, structural, or evolutionary relationships
• To convert nucleotide sequences into protein sequences without translation

Correct Answer: To identify regions of similarity that may indicate functional, structural, or evolutionary relationships

Q2. How does global alignment differ from local alignment?

• Global alignment ignores end gaps while local alignment penalizes them heavily
• Global aligns entire sequences end-to-end; local finds the highest-scoring subsequences
• Local alignment always uses a substitution matrix but global alignment does not
• Local alignment is used only for nucleotide sequences and global for proteins

Correct Answer: Global aligns entire sequences end-to-end; local finds the highest-scoring subsequences

Q3. Which algorithm is the classic method for computing optimal global pairwise alignments?

• Smith–Waterman algorithm
• Needleman–Wunsch algorithm
• BLAST heuristic
• Clustal progressive alignment

Correct Answer: Needleman–Wunsch algorithm

Q4. Which algorithm is specifically designed for optimal local alignment between two sequences?

• Needleman–Wunsch algorithm
• Progressive alignment
• Smith–Waterman algorithm
• Hidden Markov Model (HMM)

Correct Answer: Smith–Waterman algorithm

Q5. What is the affine gap penalty formula commonly used in alignment scoring?

• Gap cost = gap length × substitution matrix score
• Gap cost = gap opening penalty + (gap extension penalty × gap length)
• Gap cost = constant penalty per gap regardless of length
• Gap cost = gap opening penalty × gap extension penalty

Correct Answer: Gap cost = gap opening penalty + (gap extension penalty × gap length)

Q6. How do PAM and BLOSUM substitution matrices fundamentally differ?

• PAM is derived from conserved blocks of unrelated proteins; BLOSUM uses global alignments
• PAM is used only for nucleotides while BLOSUM is for proteins
• PAM is derived from global alignments of closely related sequences; BLOSUM is derived from conserved blocks of more diverse sequences
• There is no difference; they are identical in derivation and use

Correct Answer: PAM is derived from global alignments of closely related sequences; BLOSUM is derived from conserved blocks of more diverse sequences

Q7. What does a higher BLOSUM number (e.g., BLOSUM80) indicate about its intended use?

• It is optimized for aligning very divergent sequences
• It is optimized for aligning closely related sequences
• It should only be used for nucleotide alignments
• It ignores conservative substitutions entirely

Correct Answer: It is optimized for aligning closely related sequences

Q8. What does the E-value reported by BLAST represent?

• The exact number of homologs present in the database
• The expect value estimating how many hits one can expect to find by chance; lower is more significant
• The evolutionary distance in PAM units
• The number of mismatches in the best alignment

Correct Answer: The expect value estimating how many hits one can expect to find by chance; lower is more significant

Q9. What is the purpose of the bit score in BLAST output?

• To provide a raw unnormalized score dependent on database size
• To give a normalized score representing alignment quality independent of database size
• To count the number of gaps in the alignment
• To measure sequence complexity

Correct Answer: To give a normalized score representing alignment quality independent of database size

Q10. How does BLAST speed up database searches compared to exact dynamic programming?

• By performing global alignment instead of local alignment
• By using short exact ‘word’ matches as seeds and extending them heuristically
• By converting sequences to binary and using bitwise operations for exact alignment
• By aligning only the first 100 residues of each sequence

Correct Answer: By using short exact ‘word’ matches as seeds and extending them heuristically

Q11. What is the time and space complexity of Needleman–Wunsch dynamic programming for two sequences of lengths m and n?

• O(m + n) time and O(1) space
• O(mn) time and O(mn) space
• O(m^2 + n^2) time and O(m + n) space
• Exponential time and linear space

Correct Answer: O(mn) time and O(mn) space

Q12. How does sequence identity differ from sequence similarity?

• Identity counts identical residues; similarity includes conservative substitutions weighted by a scoring matrix
• Similarity counts identical residues; identity counts conservative substitutions
• They are interchangeable terms with no difference
• Identity measures structural similarity while similarity measures sequence length

Correct Answer: Identity counts identical residues; similarity includes conservative substitutions weighted by a scoring matrix

Q13. What is the purpose of backtracking in dynamic programming alignment algorithms?

• To fill the scoring matrix using substitution values
• To compute the E-value of the alignment
• To trace and reconstruct the optimal alignment path after filling the scoring matrix
• To convert protein alignments to nucleotide alignments

Correct Answer: To trace and reconstruct the optimal alignment path after filling the scoring matrix

Q14. What is a Position-Specific Scoring Matrix (PSSM) used for in PSI-BLAST?

• A matrix that lists only gap penalties for a sequence
• A matrix with position-specific substitution scores derived from a multiple sequence alignment used to detect distant homologs
• A simple count of nucleotide frequencies across a genome
• An alternative name for the BLOSUM matrix

Correct Answer: A matrix with position-specific substitution scores derived from a multiple sequence alignment used to detect distant homologs

Q15. What is the main principle behind progressive multiple sequence alignment (e.g., Clustal)?

• Align all sequences simultaneously using exhaustive dynamic programming
• Build the multiple alignment by aligning the most similar sequences first according to a guide tree
• Use random alignments and select the best by scoring
• Only align pairwise and report the best pair

Correct Answer: Build the multiple alignment by aligning the most similar sequences first according to a guide tree

Q16. Which multiple sequence alignment approach uses consistency information from pairwise alignments to improve accuracy?

• Progressive alignment using UPGMA only
• T-Coffee
• Needleman–Wunsch pairwise alignment
• Basic BLAST search

Correct Answer: T-Coffee

Q17. What is the rationale for performing codon-aware alignment when comparing coding DNA sequences?

• To maximize the number of gaps in the alignment
• To preserve reading frames by aligning nucleotides in triplets, often guided by translated protein alignments
• To ignore synonymous substitutions entirely
• To replace nucleotides with amino acids before alignment

Correct Answer: To preserve reading frames by aligning nucleotides in triplets, often guided by translated protein alignments

Q18. Where are gaps (indels) biologically more likely to occur in protein sequences?

• In highly conserved catalytic residues
• Randomly and uniformly across all residues
• In less conserved regions such as surface loops and inter-domain linkers
• Only at the N-terminus of proteins

Correct Answer: In less conserved regions such as surface loops and inter-domain linkers

Q19. What is a commonly used E-value cutoff in BLAST to consider hits as potentially biologically significant?

• E-value < 1 is always considered significant for homology
• E-value < 1e-5 is commonly used cutoff to consider hits significant
• E-value > 10 indicates high significance
• No cutoff is ever used; all hits are treated equally

Correct Answer: E-value < 1e-5 is commonly used cutoff to consider hits significant

Q20. What advantage do profile Hidden Markov Models (HMMs) offer for sequence alignment and database searching?

• They treat all alignment positions as equally conserved and ignore gaps
• They model position-specific residue frequencies and indel probabilities, providing sensitive detection of distant homologs
• They are equivalent to simple pairwise BLAST searches with no additional sensitivity
• They can only model nucleotide sequences, not proteins

Correct Answer: They model position-specific residue frequencies and indel probabilities, providing sensitive detection of distant homologs

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