Molecular Diagnostics and Next-Generation Sequencing Practice Flashcards

This set of vocabulary flashcards covers essential molecular diagnostic concepts, NGS quality metrics, genomic abnormalities (HRD, MSI, TMB), and laboratory quality standards based on the lecture notes.

PhiX

A well-characterised control library of bacteriophage DNA about $5.4\,kb$ long used to increase sequence diversity, improve cluster identification, and calibrate quality, especially in low-diversity libraries.

HRD

Homologous Recombination Deficiency; a functional defect in repairing DNA double-strand breaks that predicts sensitivity to PARP inhibitors and platinum-based therapies.

Paired-end sequencing

A method that reads both ends of each DNA fragment to improve alignment accuracy and aid in detecting structural variants such as gene fusions and large insertions or deletions.

Deamination

A chemical modification caused by formalin fixation that converts cytosine to uracil, leading to $C>T$ or $G>A$ artefacts during sequencing.

Tumour cellularity

The proportion of tumour cells within a specimen, essential for ensuring variant allele frequency is not overly diluted by normal cell DNA.

Macrodissection

The process of removing surrounding normal tissue to enrich tumour content and improve the sensitivity of molecular testing.

$$Q30$$

A sequencing quality metric indicating a $1$ in $1000$ probability of an incorrect base call, corresponding to $99.9\%$ accuracy.

Hybrid capture

A technology that uses labelled probes to capture genomic regions of interest before sequencing, offering uniform coverage for broad panels like OncoDEEP.

Amplicon sequencing

A sequencing method that amplifies target regions using PCR primers; it is faster and requires less DNA but is generally less flexible for broad genomic profiling.

Depth of coverage

The number of sequencing reads covering a specific genomic position; greater depth improves variant detection sensitivity.

Coverage uniformity

A metric describing how evenly sequencing reads are distributed across all target regions; poor uniformity increases the risk of false-negative results.

Validation

The process of demonstrating that a method is fit for its intended clinical purpose by establishing sensitivity, specificity, precision, accuracy, and reproducibility before patient testing.

Analytical sensitivity

The laboratory's ability to detect a variant when it is present, such as a mutation at a $2\%$ variant allele frequency.

Clinical sensitivity

The ability of a test to correctly identify patients who actually have the disease or condition of interest.

VAF

Variant Allele Frequency; the proportion of sequencing reads containing a variant allele at a specific position, which must be interpreted alongside tumour purity and ploidy.

MSI

Microsatellite Instability; repeat length changes in repetitive DNA sequences caused by a defective mismatch repair system (e.g., deficiency in MLH1, MSH2, MSH6, or PMS2).

Genomic scars

Irreversible chromosomal abnormalities such as LOH, TAI, and LST that accumulate when homologous recombination is impaired.

LOH

Loss of Heterozygosity; the loss of one parental copy of a chromosomal region, often a mechanism for inactivating tumour suppressor genes.

TAI

Telomeric Allelic Imbalance; a chromosomal imbalance extending towards the telomere, used as one of the three measures to calculate an HRD score.

LST

Large Scale State Transitions; chromosomal breakpoints caused by genomic instability, counted as part of the total HRD score calculation.

ctDNA

Circulating tumour DNA; highly fragmented DNA in the blood that typically represents a small fraction (often below $1\%$) of total cell-free DNA.

$$ISO\,15189$$

An internationally recognised framework for quality and competence in medical laboratories, ensuring validated processes and accurate, traceable results.

Repeatability

The consistency of results when the same sample is analysed under identical conditions, including the same operator, instrument, and day.

Reproducibility

The consistency of results when a sample is analysed under different conditions, such as different operators, reagent lots, or instruments.

On-target rate

The percentage of sequencing reads that map to the intended target genomic regions rather than irrelevant ones.

Duplicate reads

Multiple sequencing reads originating from the same DNA fragment, typically introduced during PCR, which artificially inflate depth and reduce library complexity.

Insert size

The length of the DNA fragment between sequencing adapters, providing information on DNA quality and library preparation.

Internal controls

Controls included within each run to verify that every stage of the assay (extraction, library prep, sequencing) performed correctly.

FISH

Fluorescence In Situ Hybridisation; the gold standard for visualising specific chromosomal abnormalities like HER2 amplification or ALK rearrangements.

SNV

Single Nucleotide Variant; a change in a single DNA base (e.g., $A > G$).

TMB

Tumour Mutational Burden; the number of non-synonymous somatic mutations per megabase ($Mb$) of coding DNA.

AMP

Anchored Multiplex PCR; a technology used by Archer that requires only one gene-specific primer, allowing for the detection of novel gene fusion partners.

Verification

The process of confirming that an already validated assay performs as expected within a specific laboratory using its own staff and equipment.

Molecular Residual Disease (MRD)

An evolving area of ctDNA testing mentioned in the 2026 ASCO guidelines for monitoring disease after treatment.

SOP

Standard Operating Procedure; a living document updated in response to new evidence, technological developments, or national guidance to maintain standardisation.