Next-generation sequencing (NGS) which is also called high-throughput or massively parallel sequencing, refers to a family of technologies that determine the order of nucleotides in DNA or RNA by reading millions of fragments simultaneously rather than one at a time, as in older Sanger sequencing. Since its commercial introduction in the mid-2000s, NGS has become the dominant sequencing approach in biomedical research and is increasingly used in clinical diagnostics, agriculture, and public-health surveillance, because it makes whole-genome and whole-exome analysis fast and comparatively affordable (Illumina, n.d^[1].; Thermo Fisher Scientific, n.d^[2].).

Most NGS workflows share four broadly similar stages, regardless of the specific instrument used.

DNA or RNA is isolated from the biological sample (blood, saliva, tissue, or other material) and its purity and concentration are checked, typically by spectrophotometry and fluorometric assays, since input quality strongly affects downstream results (iRepertoire, n.d^[3].).

The extracted nucleic acid is fragmented, and short synthetic sequences called adapters are attached to the fragment ends. RNA samples are first converted into complementary DNA (cDNA). The fragments are often amplified by PCR and, for targeted studies, may be enriched for specific genes or regions of interest before being loaded onto the sequencer (Thermo Fisher Scientific, n.d^[2].).

The prepared library is sequenced using massively parallel chemistry. Illumina's widely used "sequencing by synthesis" approach detects each nucleotide as it is incorporated into a growing DNA strand through fluorescent or, in semiconductor-based systems, electrochemical signals, generating millions of short reads simultaneously (Dunford, 2025^[4]). A newer generation of "third-generation" long-read platforms, chiefly Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), instead sequence single molecules directly: ONT measures changes in electrical current as a strand passes through a protein nanopore, while PacBio's single-molecule real-time (SMRT) method observes a DNA polymerase in action. These long-read methods produce far longer, though sometimes less individually accurate, reads that are especially valuable for resolving structural variants and repetitive genomic regions that short reads cannot span (Dante Omics, 2025^[5]; Moustakli et al, 2025^[6]).

Raw signals are converted into base calls, and reads undergo quality trimming before being aligned to a reference genome or assembled de novo. Variant-calling algorithms then identify differences from the reference, and the results are interpreted in light of the biological or clinical question being asked. Because a single sequencing run can generate terabytes of data, this stage depends on substantial computational infrastructure (Dunford, 2025^[4]); Canary Onco, n.d^[7].).

NGS underpins modern precision oncology, where comprehensive genomic profiling of tumours helps guide targeted therapy selection and monitor treatment response (Canary Onco, n.d^[7].). It is also central to rare-disease diagnosis: long-read sequencing has recently improved detection of structural variants that had long evaded short-read methods, aiding diagnosis of previously unresolved genetic disorders (Dunford, 2025^[4]). In infectious-disease surveillance, portable nanopore devices now allow near real-time pathogen identification in the field, a capability demonstrated during recent Ebola and COVID-19 outbreak responses and increasingly applied to pediatric infections and antimicrobial-resistance monitoring (Oehler et al., 2025^[8]). Metagenomic NGS is likewise expanding "One Health" surveillance of emerging zoonotic viruses at the human–animal–environment interface (Russell et al., 2025^[9]). Meanwhile, sequencing costs continue to fall: manufacturers have reported chemistry upgrades expected to reduce per-genome costs to under $300 for high-volume users, continuing a decades-long trend that has made large-scale sequencing accessible beyond specialised genome centres (Nanalyze, 2026^[10]).