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 high selectivity and enrichment for successful detection and identification.

Question 2

What are the key applications of low level mutation detection?

Answer 2

1. Somatic muts in tumour samples which are a heterogeneous mix of wt & tumour DNA (tumour itself is a heterogeneous mix of cells reducing mut level further.
2. Muts in fetus using NIPD, in which the cell-free fetal DNA load is only 3-6% of the entire DNA population (i.e. maternal DNA).
3. Heteroplasmic muts in mtDNA genomes, where mutant mtDNA is a proportion of wt mtDNA

Question 3

What is mutation ‘enrichment’ vs mutation ‘selectivity’?

Answer 3

• Selectivity of a mutation detection method refers to the selection of mutation-containing alleles among an excess of wild-type alleles
• Enrichment refers to a process that increases mutant allele concentration relative to wild-type alleles.
• The use of enrichment methods and highly selective assays is often necessary to facilitate low-level mutation detection.

Question 4

What are the different classes of mutation ‘enrichment’ methodology?

Answer 4

1. Methods differ by their ability to enrich either known or unknown muts.
2. Design of mut enrichment assays for the detection of known muts is much easier than for unknown muts as specific nucleotides can be targeted
3. As a result more methodologies exist for enrichment of known muts than unknown muts.

Question 5

What different strategies can be used to enrich and/or select for low-level mutations when the mutation is known?

Answer 5

• Preferentially destroying/blocking the wt allele
  
  • Utilised restriction enzymes
• Preferentially amplifying the variant allele
  
  • Utilises allele specific primer sequences
• Spatially separating the variant from the wt allele
  
  • Utilises specialised plates with micro-chambers or lipid droplets in emulsion

Question 6

Name various methods that use preferentially destroying/blocking of the wt allele (moderate selectivity).

Answer 6

• Restriction Endonuclease-Mediated Selective PCR (REMS-PCR)
  
  • Variant alters the sequence of a restriction enzyme (RE) site, inclusion of that RE during PCR will cause digestion of the wt amplicons (with the intact RE site) and amplification products only for the mutated allele (mutated RE site > not recognised by RE > PCR product).
• Artificial Introduction of a Restriction Site (AIRS) RFLP
  
  • If the mut does not alter a RE site, AIRS uses a modified primer that selectively binds to the wt allele and introduces a RE site into the wt PCR product during PCR > wt product of a smaller size > fragments separated by gel electrophoresis.
• Peptide Nucleic Acid (PNA)-mediated PCR and Locked Nucleic Acid (LNA)-mediated PCR
  
  • Under appropriate PCR cycling conditions, the chemically modified PNA or LNA probes specific for the wt allele will bind to the wtDNA and block primer annealing, facilitating amplification of only the variant molecules.

Question 7

Name various methods that use preferentially amplifying of the variant allele (moderate selectivity).

Answer 7

• AS-PCR (Allele-Specific PCR; key technique),
• ARMS (Amplification Refractory Mut System),
• PASA (PCR Amplification of Specific Alleles),
• PAMSA (PCR Amp of Multiple Specific Alleles),
• TaqMan or Scorpian Real-time PCR

Question 8

Name a method that has very high selectivity and exploits spatially separating the variant from the wt allele.

Answer 8

Digital PCR;

• Within a sample individual nucleic acid molecules are partitioned into separate regions.
• Each partition contains either a negative or positive reaction, i.e. “0” or “1” (i.e. digital output).
• These regions can be generated using, micro well plates, capillaries, emulsion PCR and nanofluidic chips.
• This separation allows absolute quantification (without the need for a standard curve) and a more reliable collection and sensitive measurement of nucleic acid amounts.

Question 9

Name another method with high selectivity for detection of known mutations

Answer 9

MALDI-TOF MS

Question 10

What are the two main methods of enriching/selecting for unknown mutations?

Answer 10

1. COLD-PCR (Co-amplification at lower denaturation temperature PCR) followed by a downstream detection method e.g. Sanger sequencing, MALDI-TOF
2. NGS

Question 11

What is COLD-PCR?

Answer 11

COLD_PCR is protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA.

Question 12

What is the principle of COLD-PCR?

Answer 12

• Single nucleotide mismatches will slightly alter the melting temperature (Tm) of the double-stranded DNA (Tm changes of 0.2-1.5 °C are common)
• Each dsDNA has a ‘critical temperature’ (Tc) lower than its Tm. The PCR amplification efficiency drops measurably below the Tc.
• Set an intermediate annealing temperature that allows hybridization of mutant and wildtype heteroduplexes
• Heteroduplexes are selectively denatured at the Tc
• The homo-duplex DNA will preferentially remain double stranded at the Tc and not be available for primer annealing - thus is not amplified in the extension stage.

Question 13

What are the variations of COLD-PCR?

Answer 13

• Full COLD-PCR – Enrichment of all possible muts.
  
  • Induces the formation of heteroduplexes at positions of muts.
  • By using a lower denaturation temperature during PCR, double-stranded DNA (dsDNA) containing mismatches (heteroduplexes) denature first.
  • True homoduplexes have a higher melting temperature (Tm) & denature less than heteroduplexes at the Tc; thus their amplification is relatively suppressed.
• Fast COLD-PCR – Enrichment of known Tm-reducing muts (vs. wt).
  
  • Selectively denature only variant sequences by denaturing at the Tc and then only amplify these

Question 14

What are the advantages of using COLD-PCR?

Answer 14

• Single-step method capable of enriching both known and unknown minority alleles irrespective of mutation type and position.
• Does not require any extra reagents or specialized machinery.
• Therefore the cost is not increased. Better than conventional PCR for the detection of mutations in a mixed sample. Does not significantly increase experiment run time compared to conventional PCR.

Question 15

What are the disadvantages of using COLD-PCR?

Answer 15

• Vulnerable to polymerase induced error, needs sequences less than 200bp
• All variants may not have a Tc that differs significantly from the wt
• Success depends on the sequence and position of the variant as this affects the dynamics of amplification.

Question 16

What pre-conditions are required is one is to utsilise NGS to detect low-level mutations?

Answer 16

• In order to detect low level muts by NGS, much higher read depth (deep sequencing) is needed.
• To detect rare single nucleotide variants at 0.1% requires robust processing methods, data pipeline, 10,000’s high quality parallel reads (see: Cushing et al 2013).
• Beyond this level (<0.1%) it begins to become impractical (unit cost, PCR resampling, data volume) to use NGS read depth alone to detected minority populations.

Question 17

What key factors affect the ability to utsilise NGS to detect low-level mutations?

Answer 17

1. population skewing due to differential amplification in heterogeneous mixtures
2. Polymerase mistakes resulting from base misincorporations and rearrangements due to template switching
3. Seq error is unevenly distributed throughout the genome; may be influenced by the seq context, position on the read, and molecule structure. Results in sequencing error ‘hot spots’ where the error rate can be 10x greater than the genome average. ~1% of bases are incorrectly identified, depending on the specific platform and sequence context.
4. Non-robust data analysis pipeline

Question 18

How can the performance of NGS to detect low-level mutations be improved?

Answer 18

• Unique Molecular Identifiers (UMIs): Included before amplification enabling identification of all amplicons derived from a particular starting molecule so any variation in the seq or copy number of identically tagged seq reads can be discounted as technical error. E.g. Kinde et al, reported a 20x reduction in error freq with a tagging method that is based on labeling ssDNA fragments with a primer containing a 14-bp degenerate seq.
• Duplex sequencing: Both strands of the DNA duplex are tagged and sequenced. True muts are found at the same position in both strands. In contrast, PCR or sequencing errors result in muts in only one strand and can be discounted as technical error (error rate of less 1 in a billion).
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