BN - Identifying tumour-specific mutations using Next-Generation sequencing

Question 1

Q1: What is Next-Generation Sequencing (NGS) and why is it important? (5)

Answer 1

• A second-generation DNA sequencing technology producing multiple short reads (\~100 bp) from fragmented DNA.
• Produces a consensus sequence from overlapping reads.
• Fully automated and cost-effective, now the industry standard for genome analysis.
• Enables high-throughput identification of tumour-specific mutations.
• Used in whole genome, exome, and transcriptome sequencing.

Question 2

Q2: What are the key steps in Illumina sequencing for whole-genome analysis? (6)

Answer 2

1. Fragmentation of DNA and ligation of adaptor sequences (containing primers).
2. DNA bound to solid surface via bridge amplification to form clusters.
3. Incorporation of reversible terminator nucleotides with fluorescent dyes and blocking groups.
4. Laser excitation and base detection via fluorescence.
5. Dye and blocking group are cleaved, allowing next base incorporation.
6. Repeated for each base → generates a read of up to \~300 bp.

Modified DNA polymerase and reversible terminators may introduce errors, especially near the end of reads.

Question 3

Q3: What is paired-end sequencing and why is it used? (3)

Answer 3

• Both ends of each DNA fragment are sequenced, generating two reads per fragment.
• Enables better alignment over repetitive genomic regions using known insert size.
• Produces matched files (e.g., sample_1.fq and sample_2.fq) with improved sequencing accuracy.

Question 4

Q4: What is the exome and why is it targeted in tumour sequencing? (5)

Answer 4

• The exome is the protein-coding portion of the genome (\~1–2% of total DNA).
• Contains \~85% of disease-causing mutations.
• Exome sequencing is cheaper, faster, and easier to interpret than whole-genome sequencing.
• Allows for screening of \~20,000 genes, >150,000 exons, \~50Mb of sequence.
• Used for diagnosing rare diseases, somatic cancer mutations, and molecular diagnostics.

Question 5

Q5: What are the main steps in exome sequencing analysis? (4)

Answer 5

1. Quality control (QC): Remove adaptors and low-quality sequences.
2. Alignment: Map reads to a human reference genome.
3. Variant calling: Detect mutations using a pileup of reads.
4. Annotation: Interpret mutations with reference databases (e.g., gene names, variant effect, type).

Question 6

Q6: How are mutations annotated and classified in sequencing data? (5)

Answer 6

Annotation includes: Gene name, transcript ID, exon number, nucleotide and amino acid change.

Mutation types:

• Silent – no amino acid change.
• Missense – amino acid change; may alter protein function.
• Nonsense – premature stop codon; leads to truncated protein.
• Indel – small insertion/deletion; may cause frameshifts.
• Structural mutations – large-scale changes like inversions, duplications, or translocations.

Question 7

Q7: Why is deep sequencing (e.g., 100× coverage) important in tumour analysis? (3)

Answer 7

• Ensures high confidence in mutation identification.
• Multiple overlapping paired-end reads confirm the same variant.
• Essential for detecting rare somatic mutations that define tumour specificity.

Question 8

Q8: What role does RNA-seq play in neoantigen discovery? (3)

Answer 8

• Confirms gene transcription in the tumour.
• Can sometimes validate expression of mutations at RNA level.
• RNA-seq has lower coverage and accuracy than exome sequencing, limiting its use for variant discovery.

Question 9

Q9: How are tumour-specific mutations identified? (4)

Answer 9

• Compare tumour DNA to germline DNA (e.g., from PBMCs) to find somatic mutations.
• Optional: Use RNA-seq to confirm expression.
• Helps eliminate inherited variants that are not tumour-specific.
• Identifies mutations exclusive to cancerous tissues.

Question 10

Q10: How do tissue characteristics influence mutation rates? (4)

Answer 10

• Tissues with high cell division, exposure to carcinogens (e.g., UV in skin), or chronic stress accumulate more mutations.
• Background mutation rate is not always informative once normalised for these factors.
• Driver mutations promote growth and are selected in tumours.
• Passenger mutations are random and often unique to each individual’s tumour.

Question 11

Q11: What distinguishes driver from passenger mutations in cancer? (3)

Answer 11

• Driver mutations confer a growth or survival advantage, often found in many patients.
• Passenger mutations occur randomly and accumulate without functional consequence.
• Some mutations may disrupt protein folding or function, affecting tumour biology and immune visibility.

Question 12

Q12: How can tumour-specific mutations guide immunotherapy? (3)

Answer 12

• Once mutations are identified, they are assessed for immunogenicity.
• Can be used to develop neoantigen-based T cell therapies or vaccines.
• Goal: Enable tumour-specific T cell killing with minimal off-target effects.