Next-Generation Sequencing of Cancer Genomes

Question 1

What does the central dogma of molecular biology describe?

Answer 1

The flow of genetic information within a biological system

Question 2

What are the three forms of DNA sequencing?

Answer 2

1. Whole genome sequencing
   Sequencing of the entire genome
   Can be either de novo sequencing or resequencing
2. Whole exome sequencing
   Sequencing of exomes, formed by the exons (coding regions)
3. Targeted or hot-spot sequencing
   Sequencing panel of a select number of genes

Question 3

The human genome is approximately 3.2 Giga base pairs (Gbp)

True or false

Answer 3

True

Question 4

What is required to minimise sequencing costs?

Answer 4

Careful planning of sequencing objective

Question 5

Of gene panel, whole genome and whole exome sequencing which has the highest coverage?

Answer 5

Gene panel

Question 6

What are the advantages and disadvantages of whole genome and whole exome sequencing?

Answer 6

Whole genome sequencing: low depth of coverage, high breadth of coverage

Whole exome sequencing: high depth of coverage, low breadth of coverage

Question 7

What is the difference between single and paired end sequencing?

Answer 7

Single-end (SE)- Only one end sequenced

Paired-end (PE)

• Both ends sequenced
• Precise alignment in repeat regions
• Mapping of structural variations

Question 8

1. What format is the raw sequencing format?

2. What does this contain?

Answer 8

1. FASTQ format-
   Text-based storage of biological sequences
2. Phred score-
   Quality measure for a given nucleotide

Question 9

Why is quality control needed?

Answer 9

To remove poor quality reads

• Adapter contamination
• Poor quality base calling at the start or towards the end of sequencing reads

Question 10

1. What does quality control involve?

2. What tool enables this?

Answer 10

1. Trimming of sequencing read
   - Removal of adapter sequences
   - Removal of low quality bases
2. Trimmomatic (Bolger et al. Bioinformatics. 2014

Question 11

1. After quality control what steps are pursued?

2. What is the most common tool to facilitate this?

Answer 11

Resequencing-
Requires a pre-build reference genome

Aligning sequencing reads-
Match a read to the most probable location in the reference genome

Error margin to allow for single nucleotide polymorphisms (SNPs) and single nucleotide variations (SNVs) - do not expect all sequencing reads to align perfectly to genome

2. Burrows-Wheeler Aligner (Li and Durbin Bioinformatics. 2009)

Question 12

What are variations described as?

Answer 12

A difference in a nucleotide between a sequencing read and the reference genome

Question 13

Variations are described by what?

What is the most common tool that facilities this?

Answer 13

A measure of confidence and the variant allele frequency

Mutect2

Question 14

What is a substitution?

Answer 14

Replacement of nucleotide base

Question 15

1. What are Insertions and deletions?

2. What are they commonly referred to as?

Answer 15

1. Addition or removal of nucleotide bases

2. indels

Question 16

What does Synonymous (silent) mutation result in?

Answer 16

No change in the amino acid is observed

Question 17

What is a Non-synonymous (non-silent) mutation result in?

Answer 17

Results in a change of the amino acid

• Missense: different amino acid
• Nonsense: early stop codon

Question 18

Insertions and deletions in a coding regions can result in what?

Answer 18

A frameshift If the length is not a multiple of 3

Question 19

The cancer genome can contain structural variations and single nucleotide variations

True or false

Answer 19

True

Question 20

What are structural variations?

Answer 20

Changes of large (> 1Kbp) regions of a chromosome or even whole chromosomes

Includes duplications, deletions and other rearrangements (e.g. translocations)

Question 21

What tool can be used to detect structural variations?

Answer 21

Delly (Rausch et al. Bioinformatics. 2012)

Question 22

How is structural variation identified?

What are examples?

Answer 22

By paired-end reads

1. Read pair: Distance/insert size between read one and read 2 can be informative. If insert size is short and unexpected is shorter than expected after alignment, there is a deletion and if larger than expected it is an insertion
2. Split read: Can be used to refine the exact breakpoint within genome. Refers to partial alignment of sequencing read e.g read sequencing read is split after alignment to reference genome which can indicate exact location of deletion
3. Read-depth: If there is an increase in the number of sequencing reads observed over the average coverage for a particular genomic locus, this would indicate that a duplication is present. In contrast if number of sequencing reads is lower than the average coverage a deletion has occurred

Question 23

What are driver alternations?

Answer 23

Somatic alterations that are causally implicated in oncogenesis

Confer selective advantages during the evolution of a cancer

Question 24

What are Passenger alterations?

Answer 24

Do not confer any selective advantage in cancer development

Have no functional implications

Question 25

What mutations are seen in a cancer genome?

Answer 25

Transversion: refers to a point mutation in DNA in which a single (two ring) purine (A or G) is changed for a (one ring) pyrimidine (T or C), or vice versa. Transition: A point mutation that changes a purine nucleotide to another purine (A ↔ G), or a pyrimidine nucleotide to another pyrimidine (C ↔ T). Deletion Insertion

Question 26

What structural rearrangements are seen in a cancer genome?

Answer 26

Paracentric inversion: Does not include the centromere and chromosome has two breaks in one arm of the chromosome. Reciprocal Translocation: A transfer of genetic material between homologous chromosomes. These are most commonly balanced exchanges, such that no genetic material is lost and individuals are phenotypically normal.  Copy neutral loss of heterozygosity: One of two homologous chromosomal regions is lost, but various mechanisms have ensured the presence of two identical copies of such region in the genome. As a result, the karyotype appears normal or 'copy neutral.

Question 27

What structural copy number aberrations are seen in a cancer genome?

Answer 27

Copy number gain Copy number loss

Question 28

Recurrent copy number alterations occur breast cancer What is an example

Answer 28

HER2 (ERBB2) amplification are common in HER2- positive breast cancers

Question 29

Cases with high levels of copy number alterations generally have low levels of mutations Cases with recurrent mutations have low levels of copy number alterations True or false

Answer 29

True

Question 30

All somatic mutations in a cancer genome are the result of what?

Answer 30

Multiple mutagenic processes

Question 31

The final mutational portrait provides a rich source of what information?

Answer 31

Historical or on-going processes May provide information for treatment prediction

Question 32

All cancer genomes include diploid (23n) True or false

Answer 32

False Cancer genomes often contains copy number changes

Question 33

Mutagenic processes leave distinct mutational signatures. What is one example

Answer 33

BRCA 1 and BRCA2: Base pair substitution C to A, C to G and C to T

Question 34

How does tracking evolution of the cancer genome help with selecting the best treatment?

Answer 34

Trunk- alterations in all the tumour cells , likely occurred early on in tumour development Branches- different sub clones present in tumour sample Can use data to infer which sub clones metastise and thus decide with alterations should be targeted for effective treatment

Question 35

What is the difference between linear and branched pattern of evolution?

Answer 35

Linear= Clonal changes Branched pattern of evolution= subclonal changes