7: Investigating Cancer Genomes

Question 1

What is a main motivation for large-scale cancer genome sequencing studies?

Answer 1

Identify cancer driver genes
They provide cells with a growth advantage when mutated

Question 2

Why are cancer bio banks paired with blood samples from the same patient so valuable to clinicians?

Answer 2

Shows the progress/regression of a patient, and can identify what is going on from diagnosis > flow up/relapse

Question 3

At what points through the cancer patient’s journey are blood samples taken?

Answer 3

Diagnosis
Surgery
Chemo start
Chemo end
Follow - Up
Relapse, if applicable

Question 4

Outline the steps of workflow of NGS data anaylsis

Answer 4

• Assessment of Quality
• Aligning sequences
• Identifying variants
• Annotating variants
• Visualizing NGS data

Question 5

What is NGS data analysis?

Answer 5

Next-generation sequencing

emerging technology which determines DNA/RNA sequences for whole genome or specific regions of interest

Question 6

What happens during assessment of quality in NGS data analysis?

Answer 6

• NGS reads are evaluated to remove, correct, or trim reads that don’t meet standards
• Errors include base calling errors, poor quality reads.
• This is mostly automated.

Question 7

What are NGS reads?

Answer 7

Short reads from chopping genome randomly and re-assembling them

Question 8

What happens during the aligning sequences phase of NGS data analysis?

Answer 8

Reads as aligned to reference genome, eg to GRC (genome reference consortium).

Question 9

What happens during the identifying variants phase of NGS data analysis?

Answer 9

Compares the difference between patient tumour and reference.
Sequence coverage is important in this stage as identified mutations hound be supported by multiple reads.

Question 10

What is Coverage in NGS?

Answer 10

The average number of reads that align to/cover known reference bases

Question 11

What is depth of coverage?

Answer 11

The number of reads of a given nucleotide in an experiment

Question 12

Why is coverage an important factor for variant detection?

Answer 12

• Determines wether discovery can be made with a certain degree of confidence at particular base positions
• The higher the depth of coverage, the more likely you are to find all mutants

Question 13

What are the preferred depths of coverage for normal versus cancer DNA?

Answer 13

Normal: 30x
Cancer: 60x

Question 14

Why is the preferred depth of coverage for Cancer DNA higher than for normal DNA?

Answer 14

Due to tumour heterogeneity.

Some parts of the tumour may have a mutation present, whilst other areas may not. You therefore need more coverage as normal tissues tend to be more homogenous.

Question 15

What are the 3 different groups of genomic changes in cancer?

Answer 15

• small variants (SMPs, indels. <50bp change)
• copy number alterations (amplifications, deletions)
• structural variations (inversions, translocations)

Question 16

What happens during the annotating variants of NGS data analysis?

Answer 16

Identifies disease causing variants. Annotation of SNPs and INDELs provided via computational annotation tools.

Question 17

What happens during the visualization NGS data phase of NGS data analysis?

Answer 17

Use visualization tools and genome browsers to visualize variants.
Obtain information about variants.

Question 18

What variants can we obtain by visualizing variants?

Answer 18

• Mapping quality
• Aligned reads
• Annotation information ( consequence, impact of variance, scores of annotation tools)

Question 19

How may a genome be visualization using a UCSC genome browser?

Answer 19

• Gene as a long horizontal line, and exons as small vertical lines along it.
• Arrows to denote direction of gene from promoter to 3’ end.

Question 20

What is the overall idealized pipeline Fiordland cancer genome analysis?

Answer 20

• Sequence data prep and processing (sequencing of matched tumour/normal DNA, alignment to reference genome)
• Dissect and catalogue genomic changes (Nucleotide changes, copy number alterations, structural variation)
• Consequence analysis (recurrent changes, significantly altered genes, biological pathways)

Question 21

What is the challenge of genome sequencing glioblastomas?

Answer 21

There is strong intra- and inter- tumoural heterogeneity

Question 22

What % of the mutations in cancer occur in non-coding parts of the genome?

Answer 22

98.5%

Question 23

Outline the idea of evolutionary conservation

Answer 23

• Conserved genome positions for 100 million years implies these areas are important, and have a specific function
• We can observe conserved invariant sequences across species and evolution
• We can use this to identify novel candidate driver genes