Topic overview

Start with the big picture

Sanger sequencing uses fluorescently labeled chain-terminating ddNTPs to create DNA fragments of different lengths, which are separated by capillary electrophoresis. It offers high per-base accuracy and is suited to single genes or sequence validation, but has low throughput. NGS platforms process many reactions in parallel, with distinct methods and trade-offs: Illumina uses fluorescent reversible terminators; other approaches detect light, ligation signals, pH changes, fluorescence in real time, or ionic-current changes. The lesson also introduces library preparation steps, including fragmentation, adapter ligation, size selection, and optional barcoding. Coverage depth relates to variant-detection sensitivity, while paired-end reads can support alignment across repetitive regions.