Next Generation Sequencing (NGS) - Principles, Workflow, and Applications

Vocabulary practice flashcards covering Next Generation Sequencing technology, sequencing workflows, bioinformatics analysis, and clinical variant interpretation.

Next Generation Sequencing (NGS)

A high-throughput DNA sequencing technology that allows millions of DNA fragments to be sequenced simultaneously at a lower cost per base than traditional sequencing.

Massively Parallel Sequencing

An alternate term for Next Generation Sequencing (NGS), describing its capability to conduct millions of sequencing reactions simultaneously in parallel.

Neuromuscular Disorders (NMDs)

A group of genetic diseases affecting the nerves and muscles involved in movement, with an estimated prevalence of approximately $1\text{ in }3,000$ individuals.

Monogenic Disease

A genetic disorder caused by a disease-associated variant in a single gene.

Human Genome Project

An international research initiative spanning from $1990$ to $2003$ aimed at determining the sequence of the human genome, providing the foundation for modern genomic research and NGS.

Whole Genome Sequencing (WGS)

An NGS method that sequences DNA across the entire genome, encompassing both coding and non-coding regions.

Whole Exome Sequencing (WES)

An NGS method focusing specifically on the protein-coding regions of the genome (exons), which contain approximately $85\%$ of known Mendelian disease variants.

Exome

The protein-coding regions of the genome, which represent approximately $1\text{--}2\%$ of the total human genome.

Targeted Gene Panels

An NGS approach that sequences a preselected group of genes linked to a specific clinical presentation or disease phenotype.

Sanger Sequencing

A traditional DNA sequencing technique based on chain-terminating nucleotides that typically processes one DNA region per reaction.

Sequencing by Synthesis

The sequencing mechanism used in the described NGS platforms where the incorporation of complementary nucleotides is detected in real time as DNA is synthesised.

DNA Integrity Score

A quality rating from $0$ to $4$ assessed via gel electrophoresis, where $0$ indicates no discernible high-molecular-weight DNA and $4$ indicates very little or no smearing.

DNA Fragmentation

The controlled physical breakdown of long DNA molecules into manageable segments of approximately $200\,\text{bp}$ for library preparation.

End Repair

The step in NGS sample preparation that repairs sticky, uneven fragment ends and denatures double-stranded DNA into single strands.

Target Enrichment

The selective capture and isolation of specific genomic regions of interest (such as exons or panel genes) prior to sequencing.

Baits (Probes)

Short, single-stranded DNA sequences designed to hybridise complementarily to specific genomic target regions.

Hybridisation Capture

A target enrichment strategy using biotinylated probes and streptavidin-coated magnetic beads to pull down target DNA fragments.

Biotin

A molecular affinity tag attached to complementary DNA probes that allows target-bound fragments to be captured by streptavidin.

Streptavidin

A protein coated onto magnetic beads that binds strongly to biotin tags, facilitating magnetic separation of targeted DNA.

Sequencing Adapters

Short, synthetic oligonucleotides ligated to the ends of DNA fragments that allow them to bind to sequencing beads for amplification.

Barcodes

Unique identifying nucleotide sequences integrated into adapters that allow fragments from different pooled samples to be distinguished.

Multiplexing

The practice of pooling multiple barcoded patient samples together to be sequenced within the same sequencing run.

Clonal Amplification

The generation of thousands to millions of identical copies of an individual DNA fragment attached to a bead to produce a detectable sequencing signal.

Emulsion PCR (emPCR)

A method used by the Ion Proton platform where individual DNA-bound beads are isolated inside microscopic water-in-oil droplets that serve as separate PCR chambers.

Ion Proton Detection Principle

A sequencing detection mechanism where the incorporation of a dNTP releases a hydrogen ion ($\text{H}^+$), resulting in a pH shift detected by a semiconductor well sensor.

Homopolymer

A contiguous sequence of identical base pairs (e.g., AAAA or GGGG) that produces cumulative $\text{H}^+$ signals, representing a notable error source in the Ion Proton system.

Read

The raw nucleotide sequence output determined from an individual sequenced DNA fragment.

Sequence Alignment

The bioinformatic process of matching and mapping sequenced reads against a standardised reference genome.

Variant Calling

The bioinformatic determination of positions where aligned sample reads differ from the reference genome sequence.

Variant Call Format (VCF)

The standardised text file format used in bioinformatics to record identified genetic variants and their associated metadata.

Zygosity

The state of whether an individual possesses identical or distinct alleles at a given locus.

Heterozygous (NGS Threshold)

A lecture-defined variant calling category where between $20\%$ and $80\%$ of sequenced reads at a position exhibit the variant allele.

Variant Annotation

The process of appending functional, genomic, and biological details (such as gene name, transcript, and predicted protein change) to identified variants.

Synonymous Variant

A base substitution that does not change the amino acid sequence of the resulting protein.

Missense Variant

A single nucleotide substitution that results in a codon coding for a different amino acid.

Frameshift Variant

An insertion or deletion of nucleotides not in multiples of three, which disrupts the triplet reading frame of the protein-coding sequence.

Variant Filtering

The process of removing common polymorphisms (SNPs) and non-relevant sequence changes to isolate candidate disease-causing variants.

Sequencing Artefacts

Apparent sequence deviations arising from PCR, sequencing, or data processing errors rather than true biological variation.

Penetrance

The proportion of individuals carrying a specific disease-associated genotype who express the corresponding clinical phenotype.