Lecture 6 Flashcards
What does Next generation sequencing refer to?
How do they differ from first generation technologies?
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.
What are the four NGS sequencing technologies?
Illumina (Solexa) sequencing
Roche 454 sequencing
Ion Torrent: Proton / PGM sequencing
SOLiD sequencing
Describe the Illumina (Solexa) sequencing technology
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
Describe the Roche 454 sequencing technology
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
Describe the Ion Torrent: Proton / PGM sequencing technology
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
Describe the SOLiD Sequencing technology
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
What are two long-read sequencing/ third generation sequencing technologies?
Oxford Nanopore
PacBio - SMRT DNA Sequencing
What are two features of long-read sequencing/ third generation sequencing technologies?
Single molecule sequencing
Real time sequencing
What are the advantages of long-read sequencing/ third generation sequencing technologies?
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
What are 4 applications of Next generation sequencing technologies?
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
What options comprise NGS DNA Sequencing?
Whole genome sequencing (WGS)
Whole Exome Sequencing (WES)
NGS targeted sequencing
What is the aim of Whole genome sequencing (WGS)?
What does it involve?
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
What is the aim of Whole Exome Sequencing (WES)?
What is the reasoning for this aim?
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
What is the aim of NGS targeted sequencing?
What does this use?
What does this require?
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
What are the advantages of RNA Sequencing?
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
What is ChIP Sequencing?
Chromatine Immunoprecipitation flowed by massive parallel sequencing
Describe the procedure of Bisulfite seq
What is this procedure followed by?
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.
Describe a workflow for calling variants in clinical samples?
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
What are the current applications of Sanger sequencing?
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
Define Personalised medicine
“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”
Describe Stratified medicine
“the grouping of patients based on risk of disease or response to therapy by using diagnostic tests or techniques”
What is an umbrella term for personalised and stratified medicine?
Precision Medicine
What does stratified medicine in clinical practise involve?
A clinical biomarker assessment step
How can NGS be used in the prediction of adverse drug events?
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.
What are possible effects of genetic variations in pharmacogenes that NGS can be used to predict drug response?
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
Why is identification of rare variants important in the context of stratified medicine?
Common genetic polymorphisms only explained 40% of the observed variability
Most genetic variants in drug-related genes are very rare
What are the main reasons behind the existing gap between pharmacogenomic research and its clinical implementation?
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
What is an example as to why genetic variants are important in the modulation of drug pharmacokinetics?
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
Pharmacogenomic biomarkers could provide tools to:
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
How does Warfarin demonstrate the importance of rare variants in drug response?
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
How is NGS technology used in clinical laboratories?
Gene panels
-Known target genes for approved drugs
Example – BRAF, KRAS, EGFR
WES
WGS
What is an example of the importance of epigenetic variants in stratified medicine?
Circulating tumor DNA carrying DNA methylation can be used for tumor stratification
Why is NGS application in stratified medicine important?
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
NGS is widely used in precision oncology.
What has this led to?
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.
