DNA sequencing (Sanger and NGS overview)

Sanger sequencing

• Chain-termination method: DNA polymerase copies a template using normal dNTPs plus a small proportion of fluorescently labelled dideoxynucleotides (ddNTPs).
• ddNTPs lack a 3'-OH group, so incorporation stops extension, producing fragments ending at every position.
• Each ddNTP (A, C, G, T) carries a different dye. Fragments are separated by capillary electrophoresis and read by a laser detector as an electropherogram.
• Workflow: PCR amplification, clean-up of primers and dNTPs, cycle sequencing, purification, capillary electrophoresis, analysis.
• Read length about 700–1000 bases; very accurate; gold standard for confirming variants.
• Heterozygous variants show two overlapping peaks; detection limit is roughly 15–20% variant allele fraction.

Next-generation sequencing (NGS)

• Massively parallel sequencing of millions of fragments at once.
• Library preparation: fragmentation, adapter ligation and index (barcode) addition so many samples can be pooled.
• Clonal amplification: bridge amplification on a flow cell (Illumina) or emulsion PCR on beads (Ion Torrent).
• Illumina: sequencing by synthesis with reversible dye terminators. Ion Torrent: detects pH change when a base is added.
• Long-read platforms: PacBio SMRT and Oxford Nanopore (changes in current as DNA passes through a pore), useful for structural variants and repeats.

Data analysis

• Base calling with quality scores (Phred Q30 = 99.9% accuracy), alignment to a reference genome, variant calling, annotation and classification (e.g. ACMG criteria).
• Depth of coverage (number of reads per base) determines sensitivity for low-level variants.

Quality control and pitfalls

• Include positive and negative controls; check coverage, quality scores and contamination.
• NGS findings of clinical importance are often confirmed by Sanger or another method.

Clinical use

• Inherited disease gene panels, exome and genome sequencing, tumour mutation profiling, pathogen identification and outbreak typing, TB drug resistance, non-invasive prenatal testing.