19.02.10 Low level mutation detection

Question 1

What is low level mutation detection?

Answer 1

• Detection of a variant population of DNA in a sample where the wild-type (wt) DNA greatly exceeds the variant DNA contribution
• Need to enrich sample to be able to detect it

Question 2

List 3 applications when low level mutation detection may be required

Answer 2

1) Somatic mutations in tumour samples
2) Mutations in fetus using NIPD (cffDNA)
3) Heteroplasmic mutations in mtDNA genomes

Question 3

Somatic mutations in tumour samples - why is low level detection required?

Answer 3

• Tumours are heterogeneous mix of WT and tumour DNA
• Detect mutation as early as possible from biopsy or blood containing cell free circulating tumour DNA (ctDNA)
• Assess residual disease after radiotherapy or surgery
• Disease staging/risk stratification for prognosis or personalised medicine
• Monitor therapy outcomes and cancer remission/relapse

Question 4

NIPD mutation testing - why is low level detection required?

Answer 4

• cffDNA load is only 3-6% of the entire DNA population (i.e. rest is maternal DNA)

Question 5

mtDNA testing - why is low level detection required?

Answer 5

• Mutant mtDNA can be only a small proportion of entire mtDNA

Question 6

What is enrichment?

Answer 6

• Process that increases mutant allele concentration relative to WT alleles
• Needed to increase mutant level to one where accurate analysis is possible
• Protocol is different for known and unknown mutations
• Known mutations is much easier

Question 7

Enrichment of known mutations - list techniques with moderate to high selectivity that preferentially destroy or block the WT allele

Answer 7

Most are allele-specific amplification (ASA) methodologies

1) Restriction endonuclease-mediated selective PCR (REMS-PCR)
2) Artificial introduction of a restriction site (AIRS) RFLP (restrcition fragment length polymorphism)
3) Peptide nucleic acid (PNA)-mediated PCR and locked nucleic acid (LNA)-mediated PCR

Question 8

Restriction endonuclease-mediated selective PCR (REMS-PCR)

Answer 8

If pathogenic MUT alters the sequence of a restriction enzyme (RE), then can use that enzyme during PCR to digest WT amplicon (with intact restriction site) and will only amplify products fir the mutated allele

Question 9

Artificial introduction of a restriction site (AIRS) RFLP (restrcition fragment length polymorphism)

Answer 9

If the MUT does not alter a RE site, AIRS uses a modified primer that selectively binds to WT allele and introduces a RE site into the WT PCR product during PCR. Then expose to RE, get digestion of WT product which produces smaller PCR products which can be seen on gel

Question 10

Peptide nucleic acid (PNA)-mediated PCR and locked nucleic acid (LNA)-mediated PCR

Answer 10

Under certain PCR cycling conditions, the chemically modified PNA or LNA probes specific for the WT allele will bind to the WT DNA and block primer annealing
- Only get amplification of variant molecules

Question 11

Enrichment of known mutations - list techniques with moderate to high selectivity that preferentially amplify the variant allele

Answer 11

• These use a primer with a sequence at the 3’ end that matches that of the mutant allele (but not the WT allele)
  1) AS-PCR - allele-specific PCR (key technique)
  2) ARMS (amp refractory mutation system)
  3) Taqman or Scorpain real-time PCR

Question 12

Enrichment of known mutations - list techniques with very high selectivity

Answer 12

1) RFLP-PCR based methods
2) RSM-PCR (restriction site mutation PCR)
3) Digital PCR (dPCR) - key technique

Question 13

RFLP-PCR based methods

Answer 13

Uses thermostable restriction enzymes to differentiate between WT and mutant allele

Question 14

RSM-PCR (restriction site mutation PCR)

Answer 14

Following PCR amplification of a sequence from genomic DNA, digestin with a RE generates an extra fragment (seen on gel) on either the WT allele OR the mutant allele

Question 15

Digital PCR (dPCR)

Answer 15

• Within a sample individual nucleic acid molecules are partitioned into separate regions
• Each partition contains either a negative or positive reaction
• PCR is performed from a single template molecule
• Each well is analysed for the presence of PCR products of MUT or WT sequences using florescent probes
• Separation allows absolute quantification and a more reliable collection and sensitive measurement of nucleic acid amounts
• dPCR can be used for known and unknown mutations (for known mutations allele specific fluorescent probe detection using real-time PCR; unknown mutations use NGS to sequence products)

Question 16

What techniques are used for the detection of unknown mutations?

Answer 16

1) COLD-PCR (co-amplification at lower denaturation temperature PCR) - key technique
2) NGS sequencing - key technique

Question 17

COLD-PCR (co-amplification at lower denaturation temperature PCR)

Answer 17

• Single step method - can enrich known and unknown mutations during PCR
• Advantages - single step, known and unknown MUTs, no extra reagents, no increased time
• Disadvantages - vulnerable to polymerase induced error, variants need a Tc different to WT allele
• Two types - full and fast

Question 18

FULL COLD-PCR

Answer 18

• After the first few PCR cycles, PCR programme is switched to one that after dematuration the products cross-hybidised at an intermediate temp
• As the MUT alleles are the minority, most end up in a heteroduplex with a lower Tm than the WT homoduplex
• Temp is then raised to Tc to denature the heteroduplex preferentially
• Temp is then reduced to 55 degrees to allow primers to bind, prime replication of the preferentially denatured sequences
• True homoduplexes have a higher Tm and denature less than heteroduplexes at the Tc (thus their amp is relatively suppressed)

Question 19

FAST COLD-PCR

Answer 19

• Simplier method to enrich for mutations that reduce the Tm of the WT amplicon
• Uses the Tc rather than the standard 94 degrees denaturation temp which preferentially denatures the lower Tm allele
• So selectively denature only variant sequences by denaturing at the Tc and then only amplify these

Question 20

NGS sequencing

Answer 20

Key challenges:

1) Population of skewing due to differential amplification in heterogeneous mixtures
2) Polymerase mistakes result in base misincorporations and rearrangements due to template switching
3) Much higher read depth is required to detect low level mutations
- Seq errors are unevenly distributed throughout the genomes - so get seq error ‘hot spots’ where the error rate can be 10x greater than genome average of 1%
- To detect variants at 0.1% ned robust processing methods, data pipeline, 10,000s of high quality parallel reads
- Freq less than 0.1% and its not feasible (cost, data volume)

Question 21

How can you improve sequencing accuracy in NGS? Two examples.

Answer 21

1) Exogenous tagging (spike-ins)
- Tagging before amplification enables identification of all amplicons derived from a particular starting molecule so any variation in the sequence can be discounted as technical error
2) Duplex sequencing
- Used to detect mtDNA mutations
- Both strands of the DNA duplex are tagged and sequenced
- True MUTs are found at the same position on both strands
- Errors only found on one strand and are discounted