Di Resta

Question 1

Positives of genetic testing

Answer 1

Carrier screening (eg. in pregnancy), predictive testing, identification of susceptibility genes

Question 2

Negatives of genetic testing

Answer 2

Privacy issues
Ethical testing - consanguinity
incidental findings, which can lead to psychological impact
Informed consent is needed

Question 3

Mendelian disorders

Answer 3

One causative gene for a specific gene
Eg. DMD for Duchenne

Causative relationships, because the presence of a mutation on that specific gene gives the disease phenotype

Opposite in oligogenic disorders

Question 4

Utility of genetic testing

Answer 4

1. Mendelian disorders
2. Susceptibility genes - associated with increased risk of having the disease
3. Chromosomal alterations
4. Carrier screening

Question 5

When is It possible to perform genetic testing

Answer 5

• Pre-natal : analysis of specific mutations in the fetus. Either invasive or non-invasive techniques
• Post-natal

Question 6

DNA extraction samples

Answer 6

1. Peripheral blood
2. Chorionic villi
3. Amniocytes
4. Saliva
5. Bone and teeth - rare, used in autopsies

Question 7

Genetic counseling

Answer 7

Multidisciplinary process involving patient, the referring clinician and the genetical, and the lab doctor

Evaluates clinical data, pedigrees and family history to identify the appropriate genetic test

Question 8

Genetic lab

Answer 8

Pre PCR area –> DNA is extracted, ready for amplification
PCR area
Post-PCR area –> after the amplification we have amplicons, which undergo sequencing

Question 9

Storage

Answer 9

issue of privacy - physical storages are used
Supercomputers are needed to store the terabytes

Question 10

First generation sequencing

Answer 10

PCR amplification
1. Sanger or direct sequencing (chain terminator sequencing)

highest accuracy

Question 11

Second generation sequencing

Answer 11

Library preparation (amplification) for the region of interest
1. Roche 454 (pyrosequencing)
2. Illumina (bridge amplification) - used for diagnostic testing
3. Ion torrent (label free technology)

The read length is shorter than in Sanger sequencing (200bp)

Its data accuracy depends on coverage

Library amplification depends on coverage

It Is used to perform re-sequencing, which is seuqnecing against the reference genome

Question 12

Third generation sequencing

Answer 12

No amplification needed, and NOT diagnostic
Avoids error by sequencing the entire molecule of DNA, but has the least accuracy
1. Heliscope (sequencing by synthesis)
2. Pacific bioscience (real time sequencing)
3. Nanopore technology

Question 13

Sanger sequencing

Answer 13

1. Amplification of the DNA belonging to the region of interest
2. uses chain terminators, which are linked with a fluorophore
3. Obtain different amplicons of different lengths
4. Can use an electrophoretic gel to measure the migration of the molecule based on its molecular weight

Used to obtain long reads (max. 1000 base pairs)
highest accuracy

Uses ddNTPs

Question 14

Limitations of sanger sequencing

Answer 14

Can’t be used for the diagnosis of oligogenic disorders eg. cardiomyopathies (which have a panel of 20-50 genes associated with the phenotype)

Sanger sequencing also takes around 3 months

Used only for the analysis of main causativee genes (the gene with the highest penetrance)

Thus, it has no clinical Utility in oligogenic disorders

Question 15

Coverage

Answer 15

The proportion of the genome, or the targeted region, that has been sequenced at least once

Google: It is the proportion or percentage of a genome that has been sequenced at a certain depth. It gives an idea of how much of the entire genome has been effectively read and is usually expressed as a multiple of the genome’s size.

Mean of the total number of reads that cover the target DNA

it is influenced by the number of clusters generated on the flow cell:
- the size of the target region analyzed
- the number of samples analyzed

Question 16

Ion torrent

Answer 16

Second gen. sequencing technique based on hydrogen ions

The incorporation of a nucleotide releases a H+, which is detectable at a specific voltage chain

Uses flow chips, with microwells.

The concentration of the hydrogen ion in solution changes the pH

In a homopolymeric region, if we have more than one nucleotide of the same type the change in pH is proportional to the number of nucleotides incorporated

Question 17

Illumina sequencing

Answer 17

1. Library prep
2. Bridge amplification
3. Sequencing by synthesis
4. Alignment and data analysis

The index, a short nucleotide sequence identify the molecule of DNA belonging to the specific sample. It allows the analysis of multiple samples in the same analysis without confusing them - called pooling

External to the indexes are the adaptors, which are complementary to the adaptors on the flow cell

Forms bridges

The natural competition between dNTPs reduces incorporation bias

Using a higher number of reads allows us to discriminate artifacts

Question 18

Quality score

Answer 18

Prediction of th probability of an error in base calling
- Q30: Error occurs 1/1000 times
- the lower the Q core, the higher the false positive variant calling, leading to inaccurate conclusions and higher costs of validation

Question 19

Read

Answer 19

Sequence of a single enriched fragment of sample DNA

Question 20

Read depth

Answer 20

Total number of bases sequenced and aligned at a given reference base position

Question 21

NGS data analysis

Answer 21

After sequencing, it involves forming a file that is readable

consists of three phases: Primary, secondary and tertiary

Question 22

Primary analysis

Answer 22

Image capturing and processing - fastQ file

Question 23

FastQ file

Answer 23

Master file of all the sequencing testing. Formed at the end of the primary analysis

Name of the sequencer, number of clusters, all the reads of the specific sample with all the other information about the testing

must be stored forever

Question 24

BAM file

Answer 24

Secondary analysis –> data filtering and variant calling

It involves comparing the reads with the reference genome

uses IGV software

Heterozygous variation - when 50% of the alleles of the sequence doesn’t match the allele of the reference genome

If all reads, however, are detecting one allele, this is a homozygous variant. In this case, the reference genome contains the minority allele

Problem: Correct alignment also in the presence of a pseudogene, and computation problems during alignment

Question 25

Data management after sequencing

Answer 25

FastQ -- alignment -- .bam --variant calling-- .vcf

Question 26

Vcf file

Answer 26

Tertiary analysis: Obtain a file containing all the variants, called the variant calling file List of all the variations, common and rare, allowing us to define the causative gene Operator dependent - exploit databases of all variations to detect what kind of variation it is and prioritize them

Question 27

Diagnostic odyssey

Answer 27

Sanger sequencing may introduce the patient into the diagnostic odyssey Testing --> mis-diagnosis --> ineffective treatment --> disease progression Delayed diagnosis also means higher medical expenses The clinical evaluation is STILL the first crucial starting point

Question 28

Core disease gene lists

Answer 28

For diagnostic purposes, only genes with a known causative relationship with an aberrant genotype should be included in the analysis This is to prevent incidental unwanted findings

Question 29

Clinical utility

Answer 29

To confirm a diagnostic hypothesis To analyse pro bands family members, carrying a causative mutation linked to a disease

Question 30

Incidental findings

Answer 30

Genetic variations identified by genomic sequencing but not related to the disease investigated. It means that genetic testing should aim to analyze the causative genes associated to the primary clinical questions (even if a broader panel of gene sequencing has been performed) This is especially for Sanger sequnecing

Question 31

Main issues of NGS data

Answer 31

1. Management 2. Storage 3. Security and privacy

Question 32

Classification of genetic variants

Answer 32

Follows a list of specific criteria: - Benign - Likely benign - Variation of unknown significance - Likely clinically relevant - Clinically relevant The criteria list includes: population data (so frequency of the variant), silica prediction analysis (impact on proteins)

Question 33

Phenotypic variability

Answer 33

- Incomplete penetrance - Variable expressivity

Question 34

Detection rate

Answer 34

Refers to the percentage of patient that are solved performing the genetic testing

Question 35

When do we perform genetic testing

Answer 35

To confirm a previous diagnosis that has been formulated through clinical tests This is to avoid psychological impacts on the patient - a variant of unknown significance for example but be very hard to accept from the patient

Question 36

Best model for cardiac disorders

Answer 36

Zebrafish - transparent Used to research triadic knockouts, and found that in the presence of mutated gene there is an alteration

Question 37

Allelic heterogeneity

Answer 37

Same phenotype is associated with multiple genetic variations

Question 38

BRCAPRO

Answer 38

Tool used to calculate the risk of an individual carrying a causative genetic mutation, more than 30% is considered a high risk

Question 39

MLPA

Answer 39

Multiplex Ligand probe amplification - quantitative approach to determine is there are both alleles or only one ie. if there is a deletion, the amplification of only 1 allelese will occur this can also be applied for quantification at chromosome level Steps: 1. Denaturation and hybridization 2. Ligation 3. PCR with universal primer X and Y 4. Analysis of peaks One of the two probes has a region at its 5 prime end called the Stuffer region, which is characterized by a different length

Question 40

ARMS amplification

Answer 40

Amplification refractory mutation system Based on PCR Variation amongst the sequence of primers means that the hybridization doesn't occur Used for the identification of small deletions or single nucleotide mutations To confirm the presence of the mutation detected in ARMS we perform Sanger sequencing

Question 41

Real time PCR

Answer 41

Measures PCR amplification as it occurs, allowing relative quantification Most powerful and sensitive gene analysis technique In traditional PCR, the results are collected AFTER the reaction is complete, so we can't determine the starting concentration of nucleic acids - uses dNTPs Real time PCR has a specific fluorescent probes called Taqman probes (oligonucleotides). It is complementary to the target sequence to amplify. It consists of the reporter and the quencher - if they are close to each other there is no release of fluorescence. If target is not present --> no release of the fluorescence Real time: increase of reporter fluorescence is directly proportional to the number of generated amplicons Since its relative quantification, we need a control sample with a known concentration of target DNA

Question 42

Droplet digital PCR

Answer 42

ABSLUTE quantification Requires: Primer, DNA pol, dNTPs, taqman probe Allows us to see how many molecules of mutated DNA are resent in the sample. Generates an absolute answer for the exact number of target molecules without reference to standards or endogenous control. Droplet reader detects how many droplets are positive for the wild vs mutated probes Need to apply a statistical correction - Poisson's Then calculate the fractional abundance - number of mutated molecules over the total number of molecules/droplets Has a very high sensitivity in relation to other molecular methods Used in: Organ transplantation, liquid biopsies and to detect/prevent the first stage of relapse

Question 43

Liquid vs standard biopsies

Answer 43

Standard biopsy: Localized sampling, invasive, risk, not easily obtained Liquid biopsy: Quick, minimally invasive, easily obtained - circulating nucleic acids found in body fluids can be used as well (which can arise from dying cells - powerful non invasive approach) - can be used for early detection and monitoring of disease