Sequencing

Sequencing is the laboratory process of determining the order of nucleotides in DNA or RNA. It encompasses a range of methods, from traditional Sanger sequencing to contemporary high-throughput technologies that read millions or billions of bases in a single run. The term is central to genetics, molecular biology, and genomics, enabling the mapping, comparison, and interpretation of genetic information.

Sanger sequencing, developed by Frederick Sanger and colleagues in 1977, uses chain-terminating dideoxynucleotides and capillary electrophoresis to produce accurate reads typically up to about 800–1000 bases. It remains a reference method for smaller-scale tasks and for finishing genome regions where accuracy is critical.

Modern sequencing largely refers to next-generation sequencing (NGS), a collection of methods that perform massively parallel sequencing by synthesis or ligation. Illumina platforms dominate the market and read short fragments rapidly, while other systems such as Ion Torrent, and historically Roche 454, contributed alternatives. Read lengths are usually 50–300 bases for short-read platforms, with high throughput and lower per-base cost.

Third-generation sequencing includes single-molecule approaches that produce longer reads, such as PacBio SMRT sequencing and Oxford Nanopore Technologies. These methods offer several kilobase-scale reads, real-time data, and the potential for improved assembly of complex genomes, albeit with distinct error profiles and evolving accuracy.

Applications include whole-genome, exome, transcriptome, and metagenome analyses, clinical diagnostics, and evolutionary studies. Data are analyzed with bioinformatics pipelines using formats such as FASTQ, FASTA, BAM, and VCF. Challenges include data storage, interpretation, and privacy concerns, with ongoing advances in speed, cost, and accessibility.