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Pharmacogenetics > Lecture 6 > Flashcards

Lecture 6 Flashcards

1
Q

What does Next generation sequencing refer to?

How do they differ from first generation technologies?

A

Large-scale DNA sequencing technology that allows for querying the entire genome (whole genome), the exons within all known genes (whole exome), or only exons of selected genes (target panel).

Cheaper, faster, considerably smaller sample size needed and higher accuracy. Also, because it is quicker and cheaper, it is possible to sequence more repeats than with Sanger sequencing. More repeats means greater coverage, which leads to a more accurate and reliable sequence, even if individual reads are less accurate for NGS.

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2
Q

What are the four NGS sequencing technologies?

A

Illumina (Solexa) sequencing

Roche 454 sequencing

Ion Torrent: Proton / PGM sequencing

SOLiD sequencing

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3
Q

Describe the Illumina (Solexa) sequencing technology

A

Vast number of short reads sequenced in a single stroke (100-150 bp reads are used)

Four basic steps:
library preparation, 
cluster generation,
sequencing,
data analysis
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4
Q

Describe the Roche 454 sequencing technology

A

Pioneer NGS platform launched in 2005

Can sequence much longer reads than Illumina (up to 1kb)

Steps:
Library preparation
Emulsion PCR
PTP loading
Pyrosequencing reaction
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5
Q

Describe the Ion Torrent: Proton / PGM sequencing technology

A

Method based on fact that addition of a dNTP to a DNA polymer releases an H+ion.

Steps:
DNA fragmentation (200-400bp)
Emulsion PCR
Sequence on Ion chip

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6
Q

Describe the SOLiD Sequencing technology

A

Sequencing byOligoLigation andDetection

Run requires ∼5 days and produces 3–4 Gb of sequence data

Average read length of 25–35 bp

Steps:
Library preparation
Emulsion PCR
Polony sequencing

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7
Q

What are two long-read sequencing/ third generation sequencing technologies?

A

Oxford Nanopore

PacBio - SMRT DNA Sequencing

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8
Q

What are two features of long-read sequencing/ third generation sequencing technologies?

A

Single molecule sequencing

Real time sequencing

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9
Q

What are the advantages of long-read sequencing/ third generation sequencing technologies?

A

No need for amplification

  • PCR-free
  • PCR based

Simplified and less ambiguous genome assembly

Ability to span repetitive regions (eg. transposons, satellites, gene duplications)

Identification of large structural variation (SV) and detection of epigenetic marks

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10
Q

What are 4 applications of Next generation sequencing technologies?

A

DNA sequencing
-Genome wide variant profiling

RNA-Seq
-Deep sampling of transcriptomes

ChIP-Seq
- Genome-wide mapping of
DNA-protein interactions

Bisulfite-Seq
-Detection of DNA methylation

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11
Q

What options comprise NGS DNA Sequencing?

A

Whole genome sequencing (WGS)

Whole Exome Sequencing (WES)

NGS targeted sequencing

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12
Q

What is the aim of Whole genome sequencing (WGS)?

What does it involve?

A

Aim: characterization of the entire genome of an individual

Fragments are generated from the whole genome

Identification of SNPs, CNVs, duplications, inversions, and other forms of structural variations

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13
Q

What is the aim of Whole Exome Sequencing (WES)?

What is the reasoning for this aim?

A

Aim: sequencing all protein coding regions of genes in genome

Although very small percentage of the human genome encodes for protein, about 1%, exons harbor about 85% of the mutations with large effect on disease development

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14
Q

What is the aim of NGS targeted sequencing?

What does this use?

What does this require?

A

Aim: sequence specific areas of the genome for in-depth analyses more rapidly and cost-effectively thanWGS.

Uses deep sequencing to detect known and novel variants within your region of interest

Generally requires less sample input and produces a smaller amount of data than WGS

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15
Q

What are the advantages of RNA Sequencing?

A

Provides a comprehensive view of the transcriptome

Not dependent on prior sequence knowledge

Detection of structural variations such as gene fusions and alternative splicing events

Quantifies discrete read counts aligned to a particular sequence

Increased specificity and sensitivity

Allows differentiation of isoforms

Data analysis more complex

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16
Q

What is ChIP Sequencing?

A

Chromatine Immunoprecipitation flowed by massive parallel sequencing

17
Q

Describe the procedure of Bisulfite seq

What is this procedure followed by?

A

Bisulfite conversion involves the deamination of unmodified cytosines to uracil, leaving the modified bases unchanged.

Either massive parallel sequencing methods to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.

18
Q

Describe a workflow for calling variants in clinical samples?

A

Quality control and data pre-processing quality control and alignment

Variant calling- process of comparing the aligned reads to a reference genome to identify base pair variations

Variant annotation
annotate the variants in relation to genes (e.g., within or outside a gene), codon and amino acid positions, and classify types of variants, such as nonsense, missense, exonic deletions and synonymous variants

Variant filtering

19
Q

What are the current applications of Sanger sequencing?

A

Sanger sequencing can also complement NGS in, for example: Filling ‘gaps’ in NGS data in difficult-to-sequence areas and where coverage depth is low. Re-sequencing to confirm NGS results in small but critical sections of the genome. Validating new NGS approaches by analyzing a sample with both methods

20
Q

Define Personalised medicine

A

“this term best reflects the ultimate goal of effectively tailoring treatment based on an individual’s ‘personal profile’, as determined by the individual’s genotype and phenotype data”

21
Q

Describe Stratified medicine

A

“the grouping of patients based on risk of disease or response to therapy by using diagnostic tests or techniques”

22
Q

What is an umbrella term for personalised and stratified medicine?

A

Precision Medicine

23
Q

What does stratified medicine in clinical practise involve?

A

A clinical biomarker assessment step

24
Q

How can NGS be used in the prediction of adverse drug events?

A

Inter individual variability to drug response and risk of adverse drug reaction are mostly due to polymorphisms in genes that encode proteins with functions as drug metabolises transporters and targets.

Identification of such biomarkers contributes to therapeutic success and allows for the prevention of adverse drug reactions.

25
Q

What are possible effects of genetic variations in pharmacogenes that NGS can be used to predict drug response?

A

Genetic variations in genes, including drug metabolizing enzymes, targets and borders, all used to predict drug response and risk of adverse reactions, can cause either too high or too low exposure to a drug, increase formation of toxic metabolites, increased or decreased interactions in drug target or activation of immune system which can lead to idiosyncratic drug toxicity

E.g reduced functionality enzyme lead to increased drug exposure

26
Q

Why is identification of rare variants important in the context of stratified medicine?

A

Common genetic polymorphisms only explained 40% of the observed variability

Most genetic variants in drug-related genes are very rare

27
Q

What are the main reasons behind the existing gap between pharmacogenomic research and its clinical implementation?

A

Interpretation of NGS data
-Success dependent on experiemental data and computational prediction tools

Lack of consensus guidelines

  • Different NGS platforms have different detection capacity
  • Bioinformatic pipeline validation
28
Q

What is an example as to why genetic variants are important in the modulation of drug pharmacokinetics?

A

Codeine is O-dedmethylated by CYP2D6 to its active metabolite – morphine

CYP2D6 activity determines codeine pharmacokinetics

Loss of function haplotypes:
CYP2D64, CYP2D65
and CYP2D6*6 lead to reduced morphine which affects Codeine dosage

29
Q

Pharmacogenomic biomarkers could provide tools to:

A

Avoid overdosing and subsequent ADRs

Avoid underdosing and subsequent lack of efficacy

Avoid drug use by hypersensitive individuals
Improve clinical diagnosis

Rescue drugs previously withdrawn because of ADRs

30
Q

How does Warfarin demonstrate the importance of rare variants in drug response?

A

Warfarin responses influenced by common genetic polymorphisms of the CYP genes and VKORC

However, 40% of variability in warfarin dose requirements remains unexplained by common variants and other patient specific factors

31
Q

How is NGS technology used in clinical laboratories?

A

Gene panels
-Known target genes for approved drugs
Example – BRAF, KRAS, EGFR

WES

WGS

32
Q

What is an example of the importance of epigenetic variants in stratified medicine?

A

Circulating tumor DNA carrying DNA methylation can be used for tumor stratification

33
Q

Why is NGS application in stratified medicine important?

A

Long read sequencing and pharmacogenomic variability in important genes

Accurate variant calling

Simultaneous detection of SNVs and CNVs (and epigenetic marks)

Suited for biomarker discovery in complex regions

34
Q

NGS is widely used in precision oncology.

What has this led to?

A

Potential for microsatellite instability as predictive biomarker has recently been investigated, following evidences that high levels of microsatellite instability seemed to predict a good response to immune checkpoint inhibitors – led to FDA approval of drug Pembrolizumab for microsatellite instability and cancers

TRK fusions validated as biomarkers for FDA approval of the Larotrectinib drugs.

Pharmacogenetics (16 decks)

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