Genomics and health

Question 1

Routes to gene discovery and diagnosis

Answer 1

Traditional:
- Determine mode of inheritance
- Recombination mapping using markers
- Haplotype analysis of recombinants
- Confirm by Sanger sequencing

State of the art:
- Whole exome next generation sequencing
- Lots of polymorphisms
- ‘Filter’ polymorphisms give list to candidate genes
- Confirm by Sanger sequencing

Question 2

Human genome project 1990-2003

Answer 2

• Representative human genome sequence of ~3 billion bases
• Performed using Sanger Sequencing
• Covered 92% of genome sequence

Question 3

Cost of human genome

Answer 3

Decreased dramatically over last 20 years

Becoming more feasible to use sequencing for medical research and diagnosis

Question 4

Reference genome

Answer 4

Reference genome forms the foundation of medical, function, and diversity studies

Provides common point of reference for genomic loci - Gives genes ‘addresses’, reported variants are relative to reference genome

Provides a template - guides assembly of new genomes, enables assay design and data analysis

Question 5

Genetic variations can be characterised against reference genome

Answer 5

Single nucleotide polymorphism (SNPs)

Structural variants - Deletion, Insertion, Duplication, Inversion, Translocation, Copy number variation

Question 6

Single nucleotide polymorphism (SNPs) is the most investigated variant type

Answer 6

• Ease of analysis
• Single nucleotide substitution
• Present at >1% of the population
• Roughly 4-5 million SNPs in each individual (~once every 1000 base pairs)
• Over 600 million SNPs has been reported around the world
• Single nucleotide variations (SNVs) – similar to SNPs, but without the requirement to be present at >1% of the population

Question 7

What to consider when exploring genetic variation

Answer 7

Factors to consider when choosing a sequencing technology:
- Cost - (experimental, analysis, other)
- Time - (sample preparation, run time, analysis time and sample transport.)
- Information capture - (accuracy, feature length, complex variant detection)

• Choose the appropriate tools for the intended purposes

Question 8

BRCA1 and BRCA2

Answer 8

BRCA1:
110kb/85kb in length (intron + exon)
0.006% of genome

7.8kb/10.2kb in length (exon only)
0.0005% of genome

Do we really need all the information across the genome/transcriptome?
Microarray
Enrichment/amplicon -> Gene panel sequencing
Enrichment/amplicon -> Exome sequencing

Question 9

Amplicon sequencing uses PCR to amplify the genome regions of interest

Answer 9

Design PCR primers flanking the genes of interest (e.g. genes with mutation known to associate with cancer or diseases)

Amplify the region using PCR (amplicon)

Sequence only these regions

Example: gene panel testing (Lecture 5)

Question 10

Select input + sequencing

Answer 10

DNA -> PCR/Hybridisation capture/Whole genome sequencing

PCR -> Amplicon sequencing

Hybridisation capture -> Target enrichment sequencing

Question 11

Keywords for sequencing

Answer 11

Read - The sequence corresponding to a DNA fragment

Map - Determining where the reads originated from in a genome

Depth/coverage (fold, 4X, shallow/deep sequencing)