# NSCLC Driver-Mutation Multiplex ddPCR Panel

**KRAS / EGFR / BRAF hotspots + EML4-ALK fusion -- cfDNA, Bio-Rad QX200 / QX600**

_AI Bioinformatician by Labterna  |  29 June 2026  |  GRCh38/hg38_


## 1. Targets and Clinical Context

Six clinically actionable driver alterations in non-small-cell lung cancer (NSCLC) are targeted for quantitative detection from circulating cell-free DNA (cfDNA): the KRAS codon-12 mutations G12C (c.34G>T) and G12D (c.35G>A); the EGFR sensitising mutation L858R (exon 21) and the gatekeeper resistance mutation T790M (exon 20); the BRAF V600E mutation (exon 15); and the EML4-ALK fusion variant 1 (EML4 exon 13 fused to ALK exon 20, E13;A20). KRAS G12C is the target of covalent inhibitors (sotorasib, adagrasib); EGFR L858R is a first-line TKI indication and T790M the dominant first/second-generation TKI resistance mechanism; BRAF V600E is targeted by dabrafenib+trametinib; and EML4-ALK confers sensitivity to ALK inhibitors. Tracking these from a liquid biopsy supports non-invasive genotyping and resistance monitoring.

**cfDNA constraints.** Plasma cfDNA is fragmented (modal ~167 bp); SNV amplicons are kept short (86-89 bp) to maximise recoverable template. In ddPCR every droplet is scored positive/negative, so a single false-positive droplet on a wild-type-only control is a failure -- the QC bar is the binary-droplet bar.

| Gene | Variant | Region | GRCh38 position (plus strand) | dbSNP |
| --- | --- | --- | --- | --- |
| KRAS | p.Gly12Cys | exon 2 | chr12:25245351 (C&gt;A) | rs121913530 |
| KRAS | p.Gly12Asp | exon 2 | chr12:25245350 (C&gt;T) | rs121913529 |
| EGFR | p.Leu858Arg | exon 21 | chr7:55191822 (T&gt;G) | rs121434568 |
| EGFR | p.Thr790Met | exon 20 | chr7:55181378 (C&gt;T) | rs121434569 |
| BRAF | p.Val600Glu | exon 15 | chr7:140753336 (A&gt;T) | rs113488022 |
| EML4-ALK | EML4(1-13)::ALK(20-29) | E13;A20 junction | chr2 (mRNA junction) | n/a |


## 2. Discrimination Strategy (per target)

Each SNV uses **competitive hydrolysis-probe discrimination**: one primer pair flanks the hotspot, with a mutant-allele probe (FAM) and a wild-type-allele probe (HEX) competing for the same site. This yields the allele fraction MUT/(MUT+WT) directly in one well, the WT probe doubling as the per-locus reference -- the architecture of Bio-Rad's PrimePCR ddPCR mutation assays, run at 55 degC. Allele-specific (ARMS) priming and WT-blocking LNA clamps were evaluated and rejected as the primary chemistry: ARMS gives no co-amplified WT in the same colour (allele fraction needs a separate reference and a mis-primed WT template becomes a false-positive droplet), and a WT clamp destroys the WT reference the brief asks for. Both remain available as orthogonal confirmation / sensitivity boosters.

The two KRAS codon-12 mutations share one amplicon and one WT reference probe. The **EML4-ALK fusion uses a junction-spanning probe** over the E13;A20 boundary (present only in the rearranged transcript); it requires an RNA/cDNA input arm (one-step RT-ddPCR) and is normalised to a reference gene, not a WT allele.


## 3. Panel Design -- Primers and Probes

Primers were designed with Primer3 under the Bio-Rad ddPCR Supermix buffer model (3.8 mM Mg2+, 0.8 mM dNTP, 50 mM monovalent; SantaLucia 1998 nearest-neighbour Tm). Primer Tm is the standard NN oligo Tm; probe Tm is the probe-target duplex Tm (the same NN parameters, evaluated as a heterodimer) -- confirm both on IDT OligoAnalyzer with the matching buffer before ordering. Probe sequences carry their suggested 5' fluorophore / 3' quencher in the workbook; strand was chosen to keep a non-G 5' base and the cleanest 5'-dye neighbourhood (photoinduced-electron-transfer rule). **Amplicon length:** the SNV amplicons are 86-89 bp, above the <60 bp cfDNA sweet spot, because a competitive two-primer + two-probe (WT + MUT) architecture needs that much sequence to seat both primers and the off-centred probe pair; 86 bp is the shortest that keeps a valid probe window. The fusion cDNA amplicon is 110 bp (RNA-derived input, less fragmentation-limited than cfDNA).

| KRAS G12C oligo | Role | Sequence 5'->3' | nt | Tm | GC% | Mod |
| --- | --- | --- | --- | --- | --- | --- |
| KRAS12_F | fwd | ATTAGCTGTATCGTCAAGGC | 20 | 59.5 | 45.0 | - |
| KRAS12_R | rev | TAAGGCCTGCTGAAAATGAC | 20 | 60.3 | 45.0 | - |
| KRAS12_WT | wt | AGCTGGTGGCGTAGGC | 16 | 63.9 | 68.8 | - |
| KRAS_G12C_MUT | mut | AGCTTGTGGCGTAGGC | 16 | 61.6 | 62.5 | - |


![Figure A1](../output/figures/figure_A1_KRAS_G12C.png)
_Figure A1. KRAS G12C amplicon schematic (primers, probe, variant, Tm, 3'-end)._

| KRAS G12D oligo | Role | Sequence 5'->3' | nt | Tm | GC% | Mod |
| --- | --- | --- | --- | --- | --- | --- |
| KRAS12_F | fwd | ATTAGCTGTATCGTCAAGGC | 20 | 59.5 | 45.0 | - |
| KRAS12_R | rev | TAAGGCCTGCTGAAAATGAC | 20 | 60.3 | 45.0 | - |
| KRAS12_WT | wt | AGCTGGTGGCGTAGGC | 16 | 63.9 | 68.8 | - |
| KRAS_G12D_MUT | mut | AGCTGATGGCGTAGGC | 16 | 60.9 | 62.5 | LNA |


![Figure A2](../output/figures/figure_A2_KRAS_G12D.png)
_Figure A2. KRAS G12D amplicon schematic (primers, probe, variant, Tm, 3'-end)._

| EGFR L858R oligo | Role | Sequence 5'->3' | nt | Tm | GC% | Mod |
| --- | --- | --- | --- | --- | --- | --- |
| EGFR858_F | fwd | AGGAACGTACTGGTGAAAAC | 20 | 59.8 | 45.0 | - |
| EGFR858_R | rev | TATTCTTTCTCTTCCGCACC | 20 | 59.4 | 45.0 | - |
| EGFR858_WT | wt | TTTTGGGCTGGCCAAA | 16 | 58.8 | 50.0 | - |
| EGFR858_MUT | mut | TTTTGGGCGGGCCAAA | 16 | 62.1 | 56.2 | - |


![Figure A3](../output/figures/figure_A3_EGFR_L858R.png)
_Figure A3. EGFR L858R amplicon schematic (primers, probe, variant, Tm, 3'-end)._

| EGFR T790M oligo | Role | Sequence 5'->3' | nt | Tm | GC% | Mod |
| --- | --- | --- | --- | --- | --- | --- |
| EGFR790_F | fwd | CATCTGCCTCACCTCCAC | 18 | 61.5 | 61.1 | - |
| EGFR790_R | rev | TATTGTCTTTGTGTTCCCGG | 20 | 59.9 | 45.0 | - |
| EGFR790_WT | wt | AGCTGCGTGATGAGYTGC | 18 | 65.5 | 55.6 | degen Y |
| EGFR790_MUT | mut | AGCTGCATGATGAGYTGC | 18 | 62.8 | 50.0 | LNA,degen Y |


![Figure A4](../output/figures/figure_A4_EGFR_T790M.png)
_Figure A4. EGFR T790M amplicon schematic (primers, probe, variant, Tm, 3'-end)._

| BRAF V600E oligo | Role | Sequence 5'->3' | nt | Tm | GC% | Mod |
| --- | --- | --- | --- | --- | --- | --- |
| BRAF600_F | fwd | CTGTTCAAACTGATGGGACC | 20 | 60.6 | 50.0 | - |
| BRAF600_R | rev | CTTCATGAAGACCTCACAGTA | 21 | 58.9 | 42.9 | - |
| BRAF600_WT | wt | ACAGTGAAATCTCGATGGAG | 20 | 59.4 | 45.0 | - |
| BRAF600_MUT | mut | ACAGAGAAATCTCGATGGAG | 20 | 58.8 | 45.0 | LNA |


![Figure A5](../output/figures/figure_A5_BRAF_V600E.png)
_Figure A5. BRAF V600E amplicon schematic (primers, probe, variant, Tm, 3'-end)._

| EML4-ALK v1 oligo | Role | Sequence 5'->3' | nt | Tm | GC% | Mod |
| --- | --- | --- | --- | --- | --- | --- |
| ALKfus_F | fwd | CTACTGTAGAGCCCACACC | 19 | 60.9 | 57.9 | - |
| ALKfus_R | rev | CTTGCTCAGCTTGTACTCAG | 20 | 60.3 | 50.0 | - |
| ALKfus_JUNC | junction | ACCTAAAGTGTACCGCCGGA | 20 | 65.6 | 55.0 | - |


![Figure A6](../output/figures/figure_A6_EML4_ALK_v1.png)
_Figure A6. EML4-ALK v1 amplicon schematic (primers, probe, variant, Tm, 3'-end)._


## 4. Allele Discrimination

Allele discrimination is driven by the melting-temperature gap between the matched and mismatched probe-template duplexes (probe-target duplex Tm by nearest-neighbour under the ddPCR buffer). Mutant probes are 16-20 nt with the variant base placed off-centre; BRAF V600E sits at the long (20 nt) end because its AT-rich locus needs the extra length to reach a usable Tm. Three probes whose unmodified discrimination gap is below 7 degC are flagged for a single LNA at the variant base (KRAS G12D, EGFR T790M, BRAF V600E); KRAS G12C and EGFR L858R clear the threshold unmodified. The discrimination gap (Delta-Tm) is shown for both the mutant and the WT probe -- the controlling value is the smaller of the two.

| Assay | Probe strand | nt | Variant 5'-pos | MUT Tm match/mismatch (degC) | dTm MUT / WT (degC) | Recommendation |
| --- | --- | --- | --- | --- | --- | --- |
| KRAS G12C | antisense | 16 | 5 | 61.6 / 53.6 | 8.0 / 7.8 | unmodified |
| KRAS G12D | antisense | 16 | 6 | 60.9 / 54.7 | 6.2 / 6.3 | LNA |
| EGFR L858R | sense | 16 | 9 | 62.1 / 52.6 | 9.5 / 8.7 | unmodified |
| EGFR T790M | antisense | 18 | 7 | 62.8 / 57.0 | 5.9 / 5.6 | LNA |
| BRAF V600E | antisense | 20 | 5 | 58.8 / 53.3 | 5.5 / 5.9 | LNA |


![Figure B](../output/figures/figure_B_allele_discrimination.png)

_Figure B. Mutant-probe match vs mismatch Tm._


## 5. Multiplex Compatibility QC (the make-or-break step)

Every pair among the 20 oligos (190 pairs) was screened for 3'-end-anchored cross-dimer free energy and any-alignment free energy under the ddPCR buffer, plus per-oligo hairpin, self-dimer, Tm spread and homopolymer runs. **Verdict: WARN.** The decisive inter-assay result is that **no inter-assay primer-primer 3'-end dimer is at or below -5 kcal/mol** (worst = -4.8 kcal/mol), so the six primer pairs are mutually safe to co-amplify. Primer Tm spread is 2.6 degC (58.9-61.5). 6 inter-assay interactions are flagged at the WARN level (-5.0 to -5.6 kcal/mol, each involving a probe, not two primers); no inter-assay pair reaches FAIL.

**One intra-assay interaction does reach FAIL and must be called out:** the two competitive EGFR L858R probes (WT-HEX and MUT-FAM), which are co-resident in the same well by design, form a -12 kcal/mol cross-dimer with each other (and each self-dimers at ~ -11.5 kcal/mol) -- driven by a GGCC palindrome intrinsic to the L858R locus. This mutual probe sequestration is the panel's only FAIL-severity cross-dimer. It is locus-intrinsic (both strands carry the palindrome, so it cannot be designed away by strand choice) and is addressed in the recommendations: an LNA-shortened L858R probe plus an empirical annealing-gradient check of FAM/HEX cluster separation before the assay is used.


![Figure C](../output/figures/figure_C_multiplex_matrix.png)

_Figure C. Full-panel cross-dimer matrix._

| Oligo | Flag |
| --- | --- |
| KRAS12_R | homopolymer=4(borderline); self-dimer dG=-6.8 |
| KRAS12_WT | hairpinTm=60>55; GC=69%>65 |
| KRAS_G12C_MUT | hairpinTm=60>55; GC=62%(>60,Tm-controlled) |
| KRAS_G12D_MUT | hairpinTm=60>55; GC=62%(>60,Tm-controlled) |
| EGFR858_F | homopolymer=4(borderline) |
| EGFR858_WT | homopolymer=4(borderline); self-dimer dG=-11.4 |
| EGFR858_MUT | homopolymer=4(borderline); self-dimer dG=-11.6 |
| BRAF600_R | weak 3' end (no GC in last 2; rain risk); self-dimer dG=-9.4 |
| ALKfus_F | self-dimer dG=-5.6 |
| ALKfus_JUNC | self-dimer dG=-7.3 |


## 6. Specificity

Each primer pair was run through genome-wide in-silico PCR (UCSC isPcr) against GRCh38 -- the correct specificity test for genomic cfDNA. All five SNV pairs give a single on-target product with no off-target genomic amplicon; the fusion primers (EML4 exon 13 / ALK exon 20, ~12 Mb apart in opposing genomic orientation) correctly give no genomic product, confirming the on-target product forms only from the rearranged cDNA. A focused paralog-transcriptome BLAST (RAS/RAF/ERBB families + EML4/ALK/LTK) found two benign single-oligo homologies, neither able to generate signal: the EGFR-T790M forward primer vs ERBB2 (89%, 2 mismatches) and the BRAF-V600E WT probe vs ARAF (90%, 2 mismatches). In both cases only one oligo matches the paralog, no primer pair flanks it (genomic isPcr finds no off-target product), and a hydrolysis probe generates no signal without an amplicon -- so neither is amplified or detected. "Transcriptome" specificity here is met by genome-level isPcr (every mRNA exon is also genomic) plus this targeted paralog cDNA screen, not a transcriptome-wide scan; the fusion junction probe spans an exon-exon boundary present in no normal transcript and is intrinsically transcriptome-unique.

| Assay | Method | Result |
| --- | --- | --- |
| KRAS_codon12 | isPcr vs hg38 | PASS |
| EGFR_L858R | isPcr vs hg38 | PASS |
| EGFR_T790M | isPcr vs hg38 | PASS |
| BRAF_V600E | isPcr vs hg38 | PASS |
| EML4_ALK_v1 | isPcr vs hg38 | PASS |


## 7. Common-SNP Screen (gnomAD)

Every primer and probe footprint was screened against gnomAD v4 for common germline SNPs (allele frequency >= 1%). One common variant falls under an oligo: **rs1050171 (EGFR c.2361G>A, Q787Q; gnomAD AF 0.56)** sits under the T790M probe, 8 bp from the mutation. Rather than sacrifice discrimination by shifting the probe off it, a degenerate base (Y) was placed at that probe position so the probe tolerates both alleles. The T790M probe Tm reported in Section 3 and the figures is the reference-allele match Tm (MUT 62.8 / WT 65.5 degC); in rs1050171-alt carriers the degenerate base keeps the worst-case match Tm at 60.5 / 63.1 degC -- still well above the 55 degC annealing, so binding is retained in either genotype. No common SNP sits under any primer 3' end. A T790M control gBlock carrying the rs1050171 alt (A) allele is recommended during validation to confirm the degenerate probe's tolerance.


## 8. Channel-Layout Plans

QX200 has 2 optical channels (FAM, HEX); QX600 has 6 (FAM, HEX, Cy5, Cy5.5, ROX, ATTO 590). On QX200 the six targets need ~5 wells (4 DNA + 1 RNA): genotyping 5 SNVs plus a fusion cannot collapse into one or two wells on two channels without losing per-locus allele fraction. On QX600 the panel meets the 'one-to-two reaction' goal: a single well can carry all six (each mutant on its own channel, WT references amplitude-multiplexed, fusion via one-step RT), or -- recommended for robustness -- a 2-well split that, guided by the dimer matrix, places no flagged cross-dimer pair in the same well (Well A: KRAS + BRAF + EML4-ALK; Well B: EGFR L858R + T790M). One caveat for QX200: the GC-rich KRAS codon-12 mutant probes carry two G's in their 5' neighbourhood, and on QX200 they must occupy FAM -- the most G-sensitive dye -- with no PET-optimal alternative; validate FAM amplitude there, or prefer QX600 (which moves KRAS onto the G-tolerant red channels Cy5.5/ROX).


![Figure D](../output/figures/figure_D_channel_layout.png)

_Figure D. Channel-layout plans (QX200 + QX600)._


## 9. Synthetic gBlock Controls

10 double-stranded synthetic controls (156-180 bp; amplicon + >= 30 bp flank) are provided ready to order: a mutant control per variant (positive control / LoD spike-in) and a wild-type control per amplicon (the KRAS codon-12 WT is shared). The EML4-ALK control carries the spliced E13;A20 junction and serves as a fusion-positive control directly (dsDNA, no RT needed for the control). Full sequences are in the workbook and FASTA.

| Control | Allele | Length | Carries |
| --- | --- | --- | --- |
| KRAS_G12C_MUT_gBlock | Mutant | 156 bp | p.Gly12Cys |
| KRAS_codon12_WT_gBlock | Wild-Type | 156 bp | KRAS wild-type (exon 2) |
| KRAS_G12D_MUT_gBlock | Mutant | 156 bp | p.Gly12Asp |
| EGFR_L858R_MUT_gBlock | Mutant | 156 bp | p.Leu858Arg |
| EGFR_L858R_WT_gBlock | Wild-Type | 156 bp | EGFR wild-type (exon 21) |
| EGFR_T790M_MUT_gBlock | Mutant | 158 bp | p.Thr790Met |
| EGFR_T790M_WT_gBlock | Wild-Type | 158 bp | EGFR wild-type (exon 20) |
| BRAF_V600E_MUT_gBlock | Mutant | 157 bp | p.Val600Glu |
| BRAF_V600E_WT_gBlock | Wild-Type | 157 bp | BRAF wild-type (exon 15) |
| EML4_ALK_v1_fusion_gBlock | Fusion | 180 bp | EML4 exon13 :: ALK exon20 (variant 1) |


## 10. LoB / LoD Validation Plan

Validation should follow CLSI EP17 in the binary-droplet framework:

| Step | Procedure |
| --- | --- |
| Limit of blank (LoB) | >=20 wild-type-only replicates per assay over >=3 days/operators; LoB = mean + 1.645 SD false-positive droplets. ddPCR target: 0-1 false-positive droplet per WT-only well at the locked annealing temp. |
| Limit of detection (LoD) | Spike each mutant gBlock at 5-7 decreasing copy levels into a constant WT background; >=20 replicates/level; LoD = lowest level detected in >=95% of replicates above LoB (probit/hit-rate). Established NSCLC cfDNA ddPCR assays reach ~0.1-0.5% mutant allele fraction. |
| Limit of quantitation (LoQ) | Lowest allele fraction meeting a precision target (CV <= 25%); typically at/just above LoD for ddPCR. |
| Linearity / dynamic range | Serial-dilute the mutant gBlock over >=4 logs; measured-vs-expected R^2 >= 0.98, slope 0.9-1.1; confirm no saturation of high-abundance targets in multiplex wells. |
| Annealing-temperature gradient | Validate 53-62 degC per assay and per multiplex well; lock the temperature giving zero WT-only false positives with retained mutant amplitude (rain minimised). |
| Specificity / controls | 0 false-positive droplets on WT-only gBlocks, no-template control, and BRAFP1-containing genomic DNA; re-confirm no inter-assay cross-amplification in the final multiplex well. |
| Input / fragmentation | Validate on fragmented cfDNA (sheared or clinical plasma); the fusion arm needs an RNA/cDNA input (one-step RT-ddPCR). |


### Restriction-digest compatibility

Pre-PCR restriction digestion is not required for cfDNA (already fragmented), but per design convention every ddPCR amplicon is screened at design time against the five Bio-Rad ddPCR-compatible enzymes so the assay stays usable on long templates and so the dsDNA gBlock controls and the BRAFP1-containing genomic DNA control have a fragmentation option that does not cut the amplicon. Every amplicon has at least three compatible enzymes:

| Assay | Amplicon | Compatible enzymes (no cut site) | Recommended |
| --- | --- | --- | --- |
| KRAS G12C | 86 bp | CviQI, HindIII, MseI | CviQI |
| KRAS G12D | 86 bp | CviQI, HindIII, MseI | CviQI |
| EGFR L858R | 86 bp | AluI, HindIII, MseI | AluI |
| EGFR T790M | 89 bp | CviQI, HaeIII, HindIII, MseI | CviQI |
| BRAF V600E | 87 bp | CviQI, HaeIII, HindIII, MseI | CviQI |
| EML4-ALK v1 | 110 bp | HaeIII, HindIII, MseI | HaeIII |


## 11. Recommendations and Caveats

- All Tm values are computed (SantaLucia 1998 NN under the ddPCR buffer); confirm on IDT OligoAnalyzer with the matching buffer before ordering, and treat the match/mismatch dTm values as thermodynamic estimates pending empirical cluster separation.
- Order the three LNA-flagged probes (KRAS G12D, EGFR T790M, BRAF V600E) with one LNA at the variant base if a validation gradient shows insufficient cluster separation unmodified.
- EGFR L858R is the priority risk: its two competitive probes form a -12 kcal/mol WT x MUT cross-dimer (the panel's only FAIL-severity interaction, Section 5) from a locus-intrinsic GGCC palindrome. Before use, validate FAM/HEX cluster separation empirically and adopt an LNA-shortened L858R probe if the unmodified pair under-separates; because the palindrome is on both strands, expect this to need bench optimisation, not just a strand swap.
- The EML4-ALK assay needs an RNA/cDNA input arm; for a DNA-only workflow, substitute an ALK 3'/5' imbalance assay (detects any ALK fusion but does not identify the variant). The E13;A20 junction template was reconstructed from canonical Ensembl exons and confirmed in-frame by translation; verify it against a clinical EML4-ALK variant-1 reference (e.g. a characterised cell line such as NCI-H3122) before deployment.
- This panel was designed and screened in silico; LoB/LoD, gradient, and multiplex cross-talk must be confirmed at the bench (Section 10) before clinical use.


## 12. Output Files

| File | Contents |
| --- | --- |
| NSCLC_Hotspot_ddPCR_Panel_Report.pdf | This report. |
| NSCLC_Hotspot_ddPCR_Panel_Data.xlsx | Oligos, gBlocks, multiplex QC, specificity, SNP screen, channel layout, LoB/LoD plan. |
| sequences.fasta | All 20 oligos + 10 gBlock controls. |
| figures/ | Figures A-D (PNG 300 dpi + vector PDF). |

The `scripts/` folder ships the reproducible numbered pipeline (sequence retrieval through report build).


## 13. References

1. Bio-Rad Laboratories. *Droplet Digital PCR Applications Guide* (Bulletin 6407): primer/probe design, supermix buffer, restriction-digest compatibility.
2. Bio-Rad Laboratories. Mutation Detection Multiplexing Using the QX600 ddPCR System (6-colour amplitude multiplexing).
3. A rapid, multiplex digital PCR assay for EGFR, KRAS, BRAF, ERBB2 variants and ALK/RET/ROS1/NTRK1 fusions in NSCLC. *bioRxiv* 2023.03.09.531949 / PMC10620117 (amplitude-modulation multiplexing; DNA and RNA wells separated to minimise cross-target amplification; 1% VAF DNA threshold).
4. Decousus S et al. Development of multiplex drop-off digital PCR assays for KRAS/NRAS/BRAF/PIK3CA hotspots in plasma. *J Mol Diagn* 2023 (LoD 0.08-0.18% mutant allele fraction).
5. Soda M et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. *Nature* 2007;448:561-6.
6. Sabir SR et al. EML4-ALK variants: biological and molecular properties. *Genes* 2017 (PMC5615333): ALK kinase domain encoded by exons 20-29; variant 1 = E13;A20.
7. SantaLucia J. A unified view of polymer, dumbbell and oligonucleotide DNA nearest-neighbor thermodynamics. *PNAS* 1998;95:1460-5.
8. Untergasser A et al. Primer3 -- new capabilities and interfaces. *Nucleic Acids Res* 2012;40:e115.
9. Stadhouders R et al. The effect of primer-template mismatches on 5'-nuclease assay detection. *J Mol Diagn* 2010;12:109-17.
10. Chen S et al. A genomic mutational constraint map (gnomAD v4). *Nature* 2024;625:92-100.
11. CLSI EP17-A2. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures.
12. Crockett AO, Wittwer CT. Fluorescein-labeled oligonucleotides for real-time PCR. *Anal Biochem* 2001;290:89-97 (5'-neighbourhood G-quenching of FAM).