Techniques

Question 1

What are the advantages of third generation sequencing?

Answer 1

o More accurate detection of structural variants
o Can be used to sequence DNA, RNA and methylation
o No PCR bias in sequencing
o Can be used for deciphering repetitive regions
o Can more accurately determine gene of origin in pseudogenes
o Aid the analysis and interpretation of splice variants though characterisation of alternative splicing

Question 2

What are the challenges associated with implementing third generation sequencing in clinical practice?

Answer 2

o High sequencing error rate – 10-20% PacBio, 5-20% Nanopore
o Limited bioinformatic tools available
o Quality highly correlated with sample prep and does not work on degraded DNA
o Cost of sequencing still higher than NGS

Question 3

What are the main applications of third generation sequencing in cancer?

Answer 3

o Addresses the issue of intratumour heterogeneity by identifying sub populations of cancer cells and immune cells within a single parent
o Whole genome epigenetic analysis / Direct identification of epigenetic markers
o Aids analysis and interpretation of complex cancer genomes (e.g. complex structural arrangements)
o Identification of fusion genes
o Characterisation of alternative splicing
o Liquid biopsies
o Exogenous (i.e. microbial) DNA/RNA analysis (research)
o Identification of non coding RNAs

Question 4

What are the two main third generation sequencing methods and briefly describe the chemistry’s

Answer 4

Third Generation Sequencing is provided by Pacific Biosciences (PacBio) which utilises SMRT sequencing, a method of real time sequencing based on sequencing by synthesis and Oxford Nanopore Technologies sequencing which utilises nanopores inserted into an electrical resist membrane which detects disruption in the current as DNA nucleotides are passed through.

Question 5

What techniques can be used to detect IGHV mutational status?

Answer 5

PCR followed by sequencing of the IGHV gene
NGS

Question 6

How is the IGHV mutation status determined?

Answer 6

The number of mutations in the IGHV region is compared to the most homologous germline IGHV gene found in immunoglobulin databases (e.g. GenBank) and %
difference calculated.

Question 7

What is the threshold for IGHV to be considered mutated?

Answer 7

Mutation rates greater than 2% difference from germline. <2% is considered unmutated,

Question 8

What are the components required for PCR?

Answer 8

DNA template
The sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands.

DNA polymerase
A type of enzyme that synthesises new strands of DNA complementary to the target sequence. The most commonly used is Taq Polymerase. There are two features which make it suitable for PCR. 1) It can generate new strands of DNA using a DNA template and primers. 2) they are heat resistant

Primers
Short pieces of single stranded DNA which are complementary to the target sequence. The polymerase begins synthesising new DNA from the end of the primer.

Nucleotides (dNTPs or deoxytriphosphates)
Single units of the bases A, T, G and C which are essentially building blocks for the new DNA strands.

Question 9

What enzyme is used for reverse transcription and how does it work?

Answer 9

Reverse transcriptase, a DNA polymerase enzyme that transcribes single-stranded RNA into DNA. This enzyme is able to synthesize a double helix DNA once the RNA has been reverse transcribed in a first step into a single-strand DNA

Question 10

What is the limitation of PCR?

Answer 10

PCR is an exponential reaction. PCR eventually ceases to amplify target sequence at an exponential rate and a “plateau effect” occurs, making the end point quantification of PCR products unreliable.

Question 11

What causes the “plateau effect” in a PCR reaction?

Answer 11

Inhibitors of the polymerase reaction found in the sample
Reagent limitation
Accumulation of pyrophosphate molecules
Self annealing of the accumulating product

Question 12

What does locus specific FISH identify?

Answer 12

Bind to a particular region of a chromosome to determine on which chromosome the locus/gene is located or the number of copies that exists

Question 13

What does Alphoid/Centromeric FISH identify?

Answer 13

These map to the repetitive centromeric loci found in the middle of each chromosome. They can be used to identify the number of chromosomes. Can be used with locus-specific to determine whether material missing from a chromosome.

Question 14

What does enumeration FISH identify?

Answer 14

For the detection of deletions, duplications and chromosome ploidy. Usually one set of colours is used as a ploidy control to confirm copy number (i.e. centromeric probe). A normal signal is two copies of each colour.

Question 15

What FISH probes can be used to identity a fusion?

Answer 15

Breakapart Probes - For detection of translocations when the partner gene is unknown. Probe consists of two fluorophores (Red/Green) on either side of a known breakpoint. A variety of abnormal signal patterns can be seen depending on whether there are copy changes and whether the resulting fusion has deletions.

Dual colour/Dual fusion Probes - Detection of fusions with known partner gene. Each probe spans the breakpoint and is effectively cleaved during a translocation and fused together to produce a fusion signal.

Question 16

What should be considered when choosing a molecular marker for MRD monitoring?

Answer 16

1. At relapse, some genetic loci are prone to chromosome losses or gains (e.g. FLT3-ITD/TKD)
2. The somatic origin of a given mutation may be unclear because of their recurrence in the germline (e.g RUNX1, CEBPA).
3. Clonal haematopoiesis if indeterminate potential (CHIP) associated gene mutations often persist.

Question 17

What loci are sequenced (PCR) for B cell clonality and T cell clonality ?

Answer 17

In B-cell lymphoma to date the IGH V-J and IGK gene rearrangements have been the most commonly tested loci in PCR bases clonality studies due to the small number of primers required to detect V-J rearrangements.

With respect to T lymphoproliferative disorders TRB and TRG can be analysed in parallel or consecutively and show a detection rate of nearly 100%.

Question 18

In what proportion of B-cell clonality assessments does IGH V-J and IGK sequencing not detect clonality and what follow up testing can be carried out?

Answer 18

<5% of cases. Evaluating IGH D-J and IGL targets may sometimes be useful as a second line approach.

Question 19

Why is TRD not typically used for T-cell clonality analysis?

Answer 19

Most TRD rearrangements are removed in TCRαβ-lineage T cells following rearrangement of the TRA locus which can give rise to preferential amplification and pseudoclonality.

Question 20

What are the different steps of a PCR reaction and associate temperatures?

Answer 20

PCR step 1: Denaturation - Temp 90°C to 95°C
PCR step 2: annealing - Temp 55°C to 65°C
PCR step 3: extension - Temp 70°C to 72°C

Question 21

What is long range PCR used for?

Answer 21

For diseases where the size of the fragment is important for detection long range PCR can be used. Fragile X occurs through the expansion of high GC-rich regions known as a triplet disorder (CGG). The region is too long for conventional PCR but through amplification and comparison to a ‘normal’ result the length of the expansion can be determined. Other examples of its use are in the analysis of gene with pseudogenes.

Question 22

What differences are required compared to conventional PCR to perform long range PCR?

Answer 22

1. High fidelity Taq Polymerase (e.g. Vent) - High amplification rate, low mismatch rate, high specificity and 3’ to 5’ exonuclease activity. Need to be able to synthesise for longer without errors and correct errors with proofreading.
2. PCR primers should be bigger (22-30 nucleotides) and have 50% or less GC content
3. Reaction buffer - Required the addition of MgCl2
4. DNA template - Larger amount of template DNA - should be intact and pure else polymerase wont work effectively over period of time

Question 23

What factors can play a role in non-specific binding and amplification in PCR?

Answer 23

• Primer specificity (binding to other regions, can be caused by GC rich regions)
• Primer concentration (too high causing self annealing)
• High concentration of PCR additives (too high)
• Early action of Taq polymerase (polymerisation starts too early)

Question 24

What technique can be used for PCR reactions with non-specific binding ?

Answer 24

Hot start PCR

Question 25

What is the principle of hot start PCR and which of the PCR components are different in a reaction?

Answer 25

Hot start PCR is an optimisation of PCR in which one of the reaction components will activate only during the heating step. The main objective is to limit any of the components or Taq (specifically) to prevent early amplification

Hot start PCR requires a specially modified Taq Polymerase - HotStart Taq Polymerase. Hot start taq are either enzyme linked, oligonucleotide linked or chemically modified inactive enzymes.

Question 26

What are some examples for preventing early amplification with Taq polymerase with hot start PCR?

Answer 26

1. Using the enzyme linked antibodies – polymerase is linked with a specific antibody which makes it busy and prevents DNA amplification, the antibody is temp sensitive and once the temp reaches above 70 degrees, the antibody is release from Taq and polymerisation can occur.
2. Preheating the PCR machine – The PCR machine is heated to 95 degrees before reaction preparation. All reactions are prepared on ICE and immediately transferred to the machine.
3. Freezing the PCR reaction – Another method is to freeze the PCR mixture. Once the dNTPs, primers, water and template are added to the reaction, immediately the reaction os frozen. After that Taq is added to the frozen surface and they then are added to the PCR machine.
4. Addition of Taq DNA separately – The reaction without Taq is prepared and placed in the PCR machine, once the machine achieves temp to synthesise DNA the Taq is added to the tubes
5. The use of Wax beads – Widely used and optimised for hot start is the use of wax beads. Temp-sensitive wax beads make a physical barrier between the reaction mixture and Taq. Once PCR reaches adequate temp to melt the wax, Taq mixes with the reaction mixture.
6. Highly specific oligonucleotides

Question 27

What are the two steps required prior to sequencing in NGS?

Answer 27

1) Library preparation
2) Enrichment

Question 28

What types of variants can SNP arrays detect?

Answer 28

• SNP arrays allow for analysis of DNA copy number data and the ability to detect gains, losses, loss of heterozygosity (LOH), and mosaicism on a single array.
• The have the additional advantage of being able to detect copy-neutral LOH (also called uniparental disomy). Copy-neutral LOH is a form of allelic imbalance. In copy-neutral LOH, one allele or whole chromosome from a parent is missing. This problem leads to duplication of the other parental allele. Copy-neutral LOH may be pathological.

Question 29

What are the three main components of a SNP array analysis?

Answer 29

1. An array containing immobilized allele-specific oligonucleotide (ASO) probes.
2. Fragmented nucleic acid sequences of target, labelled with fluorescent dyes.
3. A detection system that records and interprets the hybridization signal.

Question 30

What can impact the signal intensity in a SNP array?

Answer 30

· The amount of target DNA in the sample (to detect copy number), and
· The affinity between target DNA and probe (binding will occur more efficiently between sequences with 100% of matched bases than those with 90% matched bases due to a SNP mismatch)

Question 31

What are the benefits and limitations of SNP array analysis?

Answer 31

Benefits of SNP array
• No need for metaphases, utilises DNA
• potential for blood to be analysed instead of marrow
• samples could be obtained from patients too frail for bone marrow aspirate
• potential to reduce failure rates (no dividing cells required)

Drawbacks and limitations of SNP arrays:
· Unable to detect balanced rearrangements/gene fusions and whole genome ploidy changes.
· Mosaicism is not always reliable under 20-30% (depending on size and direction) i.e. so not good for Minimal residual disease (MRD) detection, or specific nucleotide mutations.

Question 32

What happens when bisulphite modification is carried out?

Answer 32

Unmethylated cytosines in DNA are converted to uracil through treatment of single stranded DNA with sodium bisulphite; this enables the differentiation between methylated and unmethylated cytosines. Bisulphite conversion is performed under acidic conditions preferentially deaminates unmethylated cytosine whilst the methyl group on 5-methylcytosone protects the amino acid group from deamination. There are several kits available for bisulphite modification.

Question 33

What methods are typically used for the testing of ctDNA and limits of detection?

Answer 33

ddPCR (targeted) - 0.001% VAF
NGS (panel) - 0.1-1% VAF

Question 34

What biological factors can impact ctDNA testing?

Answer 34

• Type of tumour
• Site of tumour
• Tumour stage (e.g. metastatic)

Question 35

What technical factors can impact ctDNA testing?

Answer 35

• Low volume of plasma yield
• Age of sample & sample storage
• Sequencing errors (NGS - False Pos)
• Technology

Question 36

Why is sample storage and extraction so important in ctDNA analysis?

Answer 36

When blood is collected in EDTA tubes and stored, the white blood cells begin to lyse and release wild type DNA into the sample in quantities many fold higher than which ctDNA is present. This makes detection of mutations or other ctDNA biomarkers more difficult.

Question 37

How can the number of cells undergoing lysis be reduced for ctDNA analysis?

Answer 37

• Store in a cell stabilisation tube (e.g. Streck tube) which can store samples for up to 7 days for ctDNA analysis
• Dont freeze a sample prior to extracting the plasma
• Process the sample as quickly as possible and within 2-4 hours if collected in a regular EDTA tube
• Never use lith hep tubes - inhibit PCR
• Perform a double centrifugation step - one to extract plasma, a second to remove debris

Question 38

What needs to be extracted from a blood sample for ctDNA analysis?

Answer 38

Plasma

Question 39

What factors can impact the quality of a tissue sample?

Answer 39

• Fixation
• Extraction Method
• Fragmentation
• Deamination on artefacts
• Tumour Heterogeneity
• Presence of Normal Tissue
• Chemsitry of assay used for testing

Question 40

What are the non-fixation variables which may affect the quality of a tissue sample?

Answer 40

• The duration of transport from patient to laboratory.
• Size of tumour specimens.
• Environmental factors such as exposure to heat, light and the concentration and age of formalin used for fixation.
• The age of blocks examined is another consideration as long-term storage in suboptimal environments can cause significant DNA damage.
• Tumour heterogeneity, and the presence of normal tissue.
• Chemistry and design strategy used for NGS (hybridisation vs amplicon).

Question 41

What are the main technologies which can be used for MRD monitoring?

Answer 41

FISH
- Cytogenetically defined rearrangements
- Low sensitivity

Quantitative RT-PCR (RNA)
- Monitors expression of leukaemic transcripts
- Most commonly monitors expression of translocation products (e.g. BCR-ABL1)
- Molecular breakpoints should be defined at diagnosis to allow monitoring
- Uses a standard curve derived from the amplification of serial dilutions of a cell line or plasmid DNA
- Allele-specific oligonucleotide (ASO)-primers required for MRD monitoring.
- Amplification conditions and sensitivity testing for each ASO primer is established by serially diluting the diagnostic material in normal mononuclear cells.
- Patient specific and relatively inexpensive

Immunological (Flow)
- Utilises specific immunological profile of the proteins on the surface of white blood cells either by fluorescently labelled antibodies or flow cytometry.
- Flow cytometry Identifies aberrant cell surface marker expression that is not seen in normal bone marrow or blood. An immunophenotype can be identified at presentation, however some relapsed disease may present with a different immunophenotype due to evolution.

QF-PCR
- Highly polymorphic markers are used to detect chimaerism in which no other markers are available.

NGS
- High sensitivity
- Specific primers for quantification not necessary
- Expensive
- Strong emphasis on bioinformatics expertise

ddPCR
- Allows for quantification without a standard curve
- More specific than RT-PCR
- No guidelines for its use in clinical setting

Question 42

What are the three main steps in Sanger sequencing?

Answer 42

1. PCR with fluorescent chain terminating dNTPs
   • The DNA sequence is used as a template for “chain termination PCR”.
   • Same mixture as normal PCR but with addition of modified nucleotides (dNTPs) called dideooxynucleotides (ddNTPs)
   • The ddNTPs lack the 3’OH group, therefore extension ceases when ddNTP is incorporated.
2. Size separation by Gel electrophoresis
   • Separation of DNA sequences by length.
   • Shorter fragments move faster than longer fragments.
3. Gel Analysis and determination of sequence
   • Reading the gel to determine the DNA sequence from smallest to largest.
   • DNA polymerase only synthesises 5’ to 3’, each terminal ddNTP will correspond to a specific nucleotide in the original sequence.

Question 43

How does Quantitative Real Time PCR (qPCR/qrt-PCR) work? (not RT-PCR = reverse transcriptase PCR)

Answer 43

Ability to monitor the amplification the target sequence.
• Reaction monitored in “real time” (rather than looking at bands on a gel or further processing it with capillary electrophoresis).
• Can be used quantitatively to measure/monitor disease.
• The reaction is placed into a Real Time machine that “watches” the reaction occur with a camera or fluorescence detector.
• There are different techniques used to allow the progress of the reaction to be monitored but they all link the amplification of DNA to the generation of fluorescence which can simply be detected with a camera during each PCR cycle.

Question 44

What are the steps of Quantitative Real Time PCR?

Answer 44

1) Denaturation

2) Annealing
• The TaqMan probe has a gene specific sequence and binds downstream of the primer.
• At the 5’ end it has a reporter dye and at the 3’ end a quencher.
• When the probe is intact, the dye and quencher are in proximity and fluorescence resonance energy transfer (FRET) occurs, preventing fluorescence emission as the quencher absorbs the fluorescenct dye from the reporter.

3) Extension
The primers are extended by DNA polymerase.

4) Cleavage
At the target site the probe will bind downstream of a primer for the region of interest - as the primer extends, the 5’ nuclease activity of Taq DNA polymerase cleaves off the reporter dye.

5)Emission
Once the reporter is separated from the quencher the signal will increase.

More reporter dye is released at each cycle and fluorescence therefore increases with the number of cycles. This reaches an exponential (log) phase then plateaus..

Question 45

How does digital droplet PCR (ddPCR) work?

Answer 45

• Sample partitioning
• Traditional PCR, a single sample offers only a single measurement, but in ddPCR, the sample is partitioned into 20,000 nanoliter-sized droplets.
• Partitioning enables the measurement of thousands of independent amplification events within a single sample.
• Involves generating droplets by mixing the aqueous sample with oil which causes the dispersion into single droplets (as opposed to Chip dPCR which involves partitioning the reaction mix over a chip).
• Each partition should contain a single template, which will either be positive or negative (fluorescent or not fluorescent).

Question 46

Through what mechanisms do ctDNA enter circulation?

Answer 46

Cell death through apoptosis and necrosis and ctDNA is also thought to arise due to active release from tumours

• DNA integrity can help to distinguish the mechanism of cfDNA release:
o Apoptosis results in smaller DNA fragments which can vary in size but have been stated to be typically around size of ~160-200 bp which is linked to the size of DNA fragment around a nucleosome and on the linker histone4,15-18.
o Necrosis results in much larger DNA fragments up to thousands of bp in length4,15-18
o Active release from tumours can result in variable sizes of DNA, between the size of those from apoptosis or necrosis.

Question 47

What methods can be used for detecting variants in ctDNA and the pros and cons?

Answer 47

• Targeted PCR based approaches include qualitative methods such as allele specific PCR, PCR using peptide nucleic acid clamps and quantitative approaches such as and ddPCR.
o Advantageous as they require less input DNA, have a high sensitivity and allow a quick turnaround time for key variants.
o Only able to look at a small set of variants

• Next generation sequencing (NGS) based approaches are becoming more appealing due to the increase in the number of potential targets genes / variants which can influence patient care. NGS approaches would allow for multiple regions of interest to be analyses as opposed to fewer targeted variants. This would perhaps offer a clear picture of the ‘clonal differences in tumour cell populations’1.
o Differences between methodologies – amplification versus hybridisation based – would need to be considered.
o Sensitivity of NGS methodologies for ctDNA have improved due to the introduction of unique molecular identifiers (UMIs)14,18. These label each fragment with a UMI during the first PCR cycle during the library preparation stage; this means that any PCR products which have derived from this fragment can be combined into a consensus sequence. This minimises PCR artefacts and errors18

Question 48

What types of variants can SNP arrays detect?

Answer 48

SNP arrays allow for analysis of DNA copy number data and the ability to detect gains, losses, loss of heterozygosity (LOH), and mosaicism on a single array.
• The have the additional advantage of being able to detect copy-neutral LOH (also called uniparental disomy). Copy-neutral LOH is a form of allelic imbalance. In copy-neutral LOH, one allele or whole chromosome from a parent is missing. This problem leads to duplication of the other parental allele. Copy-neutral LOH may be pathological.

Question 49

Describe the different phases of a PCR reaction and associated temperatures

Answer 49

• Denaturation – 94-98 C
• Primer annealing - 50-60 C
• DNA synthesis – 69-72 C
• (Repeat)

Question 50

For ctDNA analysis, how long can an EDTA be processed for?

Answer 50

Within 1 hour the plasma needs to be extracted

Question 51

For ctDNA analysis, how long can a cell stabilisation sample be processed for?

Answer 51

Arrive at lab within 48 hours, 72 hours for processing

Question 52

What are the differences between reverse transcription PCR (RT-PCR), quantitative real-time PC (qRT-PCR) and reverse transcription quantitative real-time PCR? (RT-qPCR)?

Answer 52

• Reverse transcription PCR allows the use of RNA as a template to generate complementary DNA (cDNA) using the reverse transcriptase enzyme. This can be amplified by DNA polymerase creating a double stranded cDNA feeding into the standard PCR based process.
• qPCR describes quantitative real time PCR which is the amplification of DNA in real time measured by a fluorescent probe, most commonly and intercalating dye or probe (e.g. Taqman probe) which enables quantification of the product.
• RT-qPCR refers to reverse transcription quantitative real-time PCR (RT-qPCR) which combines RT-PCR and qPCR to enable the measurement of RNA levels through the use of cDNA in a qPCR reaction and allows for the detection of gene expression changes.

Question 53

What are some reasons an RT-PCR analysis may miss a fusion variant?

Answer 53

Present but not at breakpoints covered by probes
Neoplastic cell content too long
Fusion is present but does not result in a functional product