1. Third generation sequencing is mainly characterized by:
A. Short read length
B. PCR amplification
C. Single-molecule sequencing
D. Low error rate only
Answer: C
Rationale: TGS sequences single DNA molecules without amplification.
2. Which of the following is a third-generation sequencing technology?
A. Illumina
B. Ion Torrent
C. PacBio SMRT
D. Sanger sequencing
Answer: C
Rationale: PacBio SMRT is a classic third-generation platform.
3. Oxford Nanopore sequencing works based on:
A. Fluorescence detection
B. pH change
C. Electrical current disruption
D. Radioactive labeling
Answer: C
Rationale: Nanopore detects changes in ionic current as DNA passes through pores.
4. A key advantage of third-generation sequencing is:
A. Low cost per base
B. Extremely short reads
C. Long read lengths
D. Requirement of PCR
Answer: C
Rationale: TGS produces very long reads (up to megabases).
5. PacBio sequencing is also known as:
A. SBS
B. SMRT sequencing
C. Pyrosequencing
D. Nanopore sequencing
Answer: B
Rationale: PacBio uses Single Molecule Real-Time (SMRT) sequencing.
6. In SMRT sequencing, DNA synthesis is monitored in:
A. Flow cells
B. Zero-mode waveguides
C. Microarrays
D. Capillaries
Answer: B
Rationale: ZMWs allow observation of single polymerase molecules.
7. Which enzyme is essential in PacBio SMRT sequencing?
A. Ligase
B. Restriction enzyme
C. DNA polymerase
D. RNA polymerase
Answer: C
Rationale: DNA polymerase incorporates fluorescent nucleotides.
8. Nanopore sequencing does NOT require:
A. DNA polymerase
B. Adapter ligation
C. Motor protein
D. Nanopore protein
Answer: A
Rationale: Nanopore sequencing does not use polymerase-based synthesis.
9. One major limitation of third-generation sequencing is:
A. Inability to sequence RNA
B. High raw error rate
C. Short read length
D. Need for cloning
Answer: B
Rationale: TGS has higher raw error rates compared to NGS.
10. Which error type is most common in nanopore sequencing?
A. Substitution
B. Deletion
C. Insertion and deletion
D. Frameshift only
Answer: C
Rationale: Nanopore sequencing mainly produces indel errors.
11. Third-generation sequencing is especially useful for:
A. SNP genotyping only
B. Short amplicon analysis
C. Genome assembly
D. Microarray analysis
Answer: C
Rationale: Long reads improve de novo genome assembly.
12. Which sequencing platform can generate reads longer than 100 kb?
A. Illumina HiSeq
B. Ion Proton
C. Oxford Nanopore
D. Sanger sequencing
Answer: C
Rationale: Nanopore sequencing can generate ultra-long reads.
13. A major application of TGS is:
A. Gene expression microarrays
B. Structural variation detection
C. ELISA
D. Western blotting
Answer: B
Rationale: Long reads are ideal for detecting large structural variants.
14. Which molecule can be sequenced directly using third-generation sequencing?
A. DNA only
B. RNA only
C. Protein
D. DNA and RNA
Answer: D
Rationale: TGS platforms can sequence both DNA and RNA directly.
15. Direct RNA sequencing is possible using:
A. Illumina
B. PacBio only
C. Oxford Nanopore
D. Sanger sequencing
Answer: C
Rationale: Nanopore allows direct RNA sequencing without cDNA synthesis.
16. Which feature distinguishes TGS from NGS?
A. Use of fluorescent dyes
B. Amplification-free sequencing
C. Paired-end reads
D. Cluster generation
Answer: B
Rationale: TGS avoids PCR amplification.
17. SMRT sequencing detects nucleotides based on:
A. Electrical resistance
B. Color-coded fluorescent tags
C. pH changes
D. Radioactive signals
Answer: B
Rationale: Each nucleotide has a distinct fluorescent label.
18. The circular DNA template used in PacBio is called:
A. Linear adapter
B. Hairpin adapter
C. SMRTbell template
D. Plasmid vector
Answer: C
Rationale: SMRTbell templates enable continuous sequencing.
19. Consensus accuracy in PacBio improves due to:
A. PCR amplification
B. Circular consensus sequencing
C. Short read correction
D. Random priming
Answer: B
Rationale: Multiple passes over the same template increase accuracy.
20. Which of the following is NOT a third-generation sequencing platform?
A. PacBio RS II
B. MinION
C. Sequel II
D. Illumina NovaSeq
Answer: D
Rationale: NovaSeq is a second-generation (NGS) platform.
21. Third-generation sequencing is highly useful in studying:
A. Point mutations only
B. Epigenetic modifications
C. Protein structure
D. Cell signaling
Answer: B
Rationale: TGS can detect DNA methylation directly.
22. DNA methylation detection in TGS is possible because:
A. Bisulfite treatment is used
B. PCR bias is introduced
C. Signal kinetics are altered
D. Restriction enzymes are applied
Answer: C
Rationale: Modified bases alter signal patterns during sequencing.
23. Nanopore sequencing reads DNA in which direction?
A. 5′ to 3′ only
B. 3′ to 5′ only
C. Bidirectional
D. Random direction
Answer: A
Rationale: DNA passes through nanopores in the 5′ to 3′ direction.
24. Which protein forms the nanopore in ONT devices?
A. Taq polymerase
B. α-hemolysin or similar
C. Reverse transcriptase
D. Ligase
Answer: B
Rationale: Biological nanopores like α-hemolysin are used.
25. One major advantage of MinION is:
A. Extremely low error
B. Large laboratory setup
C. Portability
D. High reagent cost
Answer: C
Rationale: MinION is portable and field-deployable.
26. Third-generation sequencing reduces bias mainly because:
A. It uses short reads
B. It avoids PCR
C. It uses adapters
D. It uses clustering
Answer: B
Rationale: PCR-free sequencing avoids amplification bias.
27. Which genome feature is best resolved by long reads?
A. Single nucleotide polymorphisms
B. Tandem repeats
C. Small indels only
D. Restriction sites
Answer: B
Rationale: Long reads span repetitive regions.
28. Read length in third-generation sequencing is limited mainly by:
A. Polymerase speed
B. DNA fragment length
C. Optical resolution
D. Flow cell size
Answer: B
Rationale: Longer DNA fragments produce longer reads.
29. Which statement about TGS accuracy is correct?
A. Raw accuracy is always higher than NGS
B. Raw accuracy is lower but improves with consensus
C. Accuracy cannot be improved
D. Errors are random and uncorrectable
Answer: B
Rationale: Consensus sequencing greatly improves accuracy.
30. The data output of nanopore sequencing is:
A. Fluorescent images
B. pH curves
C. Electrical current traces
D. Autoradiographs
Answer: C
Rationale: Nanopore generates ionic current signals.
31. Third-generation sequencing is most suitable for:
A. Small targeted panels only
B. Whole-genome sequencing
C. Microarray analysis
D. Protein sequencing
Answer: B
Rationale: Long reads are ideal for whole-genome analysis.
32. Which sequencing generation allows real-time data analysis?
A. First
B. Second
C. Third
D. None
Answer: C
Rationale: TGS provides real-time sequencing output.
33. SMRT sequencing occurs in real time because:
A. DNA is amplified rapidly
B. Nucleotides are pre-labeled
C. Fluorescence is detected during incorporation
D. Images are processed later
Answer: C
Rationale: Signals are captured as nucleotides are incorporated.
34. Which application benefits most from TGS?
A. SNP chip design
B. Metagenome assembly
C. PCR primer design
D. Western blotting
Answer: B
Rationale: Long reads improve metagenomic assemblies.
35. Nanopore sequencing devices belong to:
A. Solid-state pores only
B. Biological pores only
C. Hybrid nanopores
D. Optical pores
Answer: B
Rationale: ONT primarily uses biological nanopores.
36. Which sequencing step is eliminated in TGS compared to NGS?
A. Adapter ligation
B. Base calling
C. Library amplification
D. Quality control
Answer: C
Rationale: TGS avoids PCR amplification.
37. Third-generation sequencing enables phasing because:
A. It uses short reads
B. Reads span multiple variants
C. It uses paired-end reads
D. It uses SNP arrays
Answer: B
Rationale: Long reads capture haplotypes directly.
38. Which sequencing technology can be used in remote locations?
A. Illumina MiSeq
B. PacBio Sequel
C. Oxford Nanopore MinION
D. Sanger sequencer
Answer: C
Rationale: MinION is portable and laptop-powered.
39. In TGS, higher coverage mainly improves:
A. Read length
B. Error correction
C. Library preparation
D. Sequencing speed
Answer: B
Rationale: High coverage improves consensus accuracy.
40. Which of the following is a drawback of long reads?
A. Difficulty in assembly
B. Higher computational needs
C. Limited genome coverage
D. Short read bias
Answer: B
Rationale: Long-read data requires more computational resources.
41. Which sequencing platform allows adaptive sampling?
A. Illumina
B. Ion Torrent
C. PacBio
D. Oxford Nanopore
Answer: D
Rationale: ONT can selectively sequence target regions in real time.
42. Third-generation sequencing is best described as:
A. Low-throughput, high-accuracy
B. High-throughput, short-read
C. Long-read, single-molecule
D. Clone-based sequencing
Answer: C
Rationale: TGS combines long reads with single-molecule detection.
43. Which base modification can be detected directly by TGS?
A. SNPs only
B. DNA methylation
C. Protein phosphorylation
D. RNA editing only
Answer: B
Rationale: Modified bases alter signal kinetics.
44. Nanopore sequencing library preparation is generally:
A. Very complex
B. Time-consuming
C. Rapid and simple
D. Impossible without PCR
Answer: C
Rationale: ONT offers rapid library prep kits.
45. Which sequencing generation best resolves repetitive genomic regions?
A. First
B. Second
C. Third
D. None
Answer: C
Rationale: Long reads span repetitive sequences.
46. Third-generation sequencing reduces GC bias because:
A. It uses GC-rich primers
B. It avoids amplification
C. It uses special buffers
D. It fragments DNA randomly
Answer: B
Rationale: PCR-free sequencing minimizes GC bias.
47. Which of the following best defines SMRT?
A. Synthetic Molecular Read Technology
B. Single Molecule Real-Time
C. Short Molecular Read Technology
D. Sequencing by Molecular Reaction
Answer: B
Rationale: SMRT stands for Single Molecule Real-Time sequencing.
48. Which sequencing platform produces kinetic data useful for epigenetics?
A. Illumina
B. Ion Torrent
C. PacBio
D. Sanger
Answer: C
Rationale: PacBio records polymerase kinetics.
49. Third-generation sequencing is especially important in:
A. Genome finishing
B. Restriction mapping
C. Northern blotting
D. ELISA
Answer: A
Rationale: Long reads close gaps in draft genomes.
50. The main contribution of third-generation sequencing to genomics is:
A. Lower cost than all methods
B. Elimination of bioinformatics
C. Accurate short reads
D. Long-read, single-molecule sequencing
Answer: D
Rationale: TGS revolutionized genomics with long-read, single-molecule data.
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