Lecture 4: Genomics and Health Part 1

Question 1

What sequencing method was used for the Human Genome Project?

Answer 1

Sanger sequencing

Question 2

What percentage of the human genome was sequenced by the Human Genome Project?

Answer 2

92%

Question 3

What sequencing method was used to resolve the remaining 8% of the human genome that wasn’t identified by the HGP? What was this 8% found to be?

Answer 3

Long-read next generation sequencing

8% was found to be duplication and repetitive regions

Question 4

The complete human genome was described in which year (1) and by which consortium (2)?

Answer 4

(1) 2022

(2) the Telomere-to-Telomere (T2T) consortium

Question 5

What is one of the major pros of NGS over sanger sequencing?

Answer 5

Cheaper - thousands reads sequenced at one time

Question 6

What is a reference genome and why is it useful?

Answer 6

A reference genome is a standard representation of the human genome

Useful as it forms the foundation of studies - provides a common point for genomic loci and provides a template.
Genetic variations can be characterised against the reference genome

Question 7

What are the two main genetic variations that can be investigated using genome sequencing?

Answer 7

1. Single nucleotide polymorphisms (SNPs)
2. Structural variants (E.g. deletion, insertion, duplication, inversion, translocation, copy number variation)

Question 8

True or false: structural variations are more easily detected with short-read sequencing techniques?

Answer 8

False: more easily detected with long-read sequencing techniques

Question 9

What are SNPs and why are they often investigated? (3 points)

Answer 9

Single nucleotide substitutions that vary compared to the reference genome

They are easily analysed and identified with genome sequencing

They are present at more than > 1% of the population (common)

Question 10

What is the difference between a SNP and a single nucleotide variation (SNV)?

Answer 10

SNPs are common at >1% of the population and each individual has roughly 4-5 million SNPs, where as SNVs are less common and not present at >1% of the population. SNVs refer more to the single nucleotide changes that occur with mutation.

Question 11

What is a haplotype?

Answer 11

the arrangement of SNPs on a chromosome

Question 12

What is a haplotype block?

Answer 12

A block on a single chromosome containing SNPs that are associated and tend to be inherited together because they are close to each other and recombination between these variants are rare resulting in only a few (4-6) alternative haplotypes for each block

Question 13

True or false: a disease susceptibility allele and a marker SNP are often in the same haplotype block?

Answer 13

True (although the SNP marker is not necessarily the cause of the disease susceptibility)

Question 14

What are the three main factors to consider when choosing a sequencing technology?

Answer 14

1. Cost
2. Time (sample prep, run time, sample transport)
3. Information capture (accuracy, complex variant detection, feature length)

Question 15

What are the two main types of genetic information that are sequenced?

Answer 15

1. DNA (Genome, exome)
2. RNA (transcriptome)

Question 16

What does depth mean in terms of sequencing?

Answer 16

the number of times a sequencing read covers a specific region of the genome

Question 17

Describe the steps of most DNA sequencing methods
1. preparation
2. sequencing
3. post-sequencing

Answer 17

1. fragmentation of DNA (physical, chemical, enzymatic)
2. sequence individual fragments (reads)
3. assemble reads through overlaps and map the reads to the reference genome

Question 18

Why does the read-length of a sequencing method matter?

Answer 18

1. longer reads are easier to assemble
2. short-read sequences are more accurate

Question 19

How can sequencing costs be reduced and sensitivity increased?

Answer 19

Filtering sequencing inputs (E.g. amplicon sequencing using PCR to amplify only sequence of interest (amplicons) and enrichment/depletion such as removing introns)

Microarray

Question 20

When might you want to use amplicon sequencing?

Answer 20

When you know the genomic loci you are interested in sequencing, you can amplify these specific regions using PCR and sequence only the amplicons to reduce cost and achieve the same depth.

Question 21

When might you use target enrichment for sequencing methods?

Answer 21

Target enrichment allows more targets to be enriched at once compared to amplicon sequencing
- for example: target enrichment of the exons (account for about 85% of known disease-related variants) in exome sequencing to reduce the cost

Question 22

Why might target enrichment/depletion be used in transcriptomics?

Answer 22

Poly-A-selection to enrich mRNA (protein coding transcripts)

Ribodepletion to remove the majority of rRNA (can look at tRNA, lncRNA as well as mRNA without large amounts of unnecessary rRNA)

Question 23

Give three examples of hybridisation-based target enrichment methods

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Answer 23

1. Microarray hybridisation
2. In Solution hybridisation
3. Molecular Inversion Probes

Question 24

Why might microarray be used in genomics?

Answer 24

It is a hybridisation method that can be used for target enrichment as well as quantification of a known DNA sequence in a sample

It is easier to analyse than sequencing data and costs less

Good at detecting copy number variations as can be quantified

Question 25

What are three possible uses of microarray in genomics?

Answer 25

Can be used for:
- Array-based comparative genomic hybridisation (aCGH) to detect copy number variations
- single-nucleotide polymorphism
- Transcriptomics

Question 26

What three controls may be used when comparing generic makeup of an individual

Answer 26

1. compare DNA of disease cell (E.g. cancer) with healthy cell of individual
2. compare DNA of disease individual with unaffected family members (E.g. in paediatric disorders to determine if its de novo mutation or familial genetic disorder)
3. Compare DNA of individual with control population that doesn’t have that phenotype

Question 27

What is HapMap?

Answer 27

HapMap is a human haplotype map that described the chromosome regions with sets of strongly associated SNPs from geographically diverse cohort

Question 28

Why is it important to expand the genome datasets (large-scale multi-omic projects)?

Answer 28

• reduce cost in long run
• identify common features for different ethinic groups and generate different reference genomes
• allow more statistically accurate conclusions to be made about SNP associations
• identify more genomic variations that will improve identification of disease causing genes.
• multi-omic (E.g. genomic and transcriptomic sequencing) studies allows us to see impact of genomic variation on encoded protein

Question 29

What are genome-wide association studies (GWAS)?

Answer 29

Uses SNPs to determine the association between a disease trait and a SNP

Question 30

What are the major challenges in variant discovery and diagnosis

Answer 30

Biased data collection:
- ethnicity (reference genome predominantly of European ancestry means there is poorer ability to identify contributing genetic variants in non-European populations)
- Participation bias (recruitment of participants is skewed towards certain demographics - ethnicity, age, gender, socio-economic status, education, etc)

Question 31

The boundaries between haplotype blocks represents hot spots for what?

Answer 31

Recombination

Question 32

What is linkage disequilibrium?

Answer 32

particular alleles of different SNPs within a haplotype block tend to be inherited together