In next-generation sequencing (NGS), the 'sequencing by synthesis' approach used by Illumina platforms employs reversible terminator chemistry. What differentiates this from Sanger sequencing in terms of scale?

• A Illumina reads one molecule per run, while Sanger reads millions simultaneously
• B Illumina uses ddNTPs as permanent terminators like Sanger
• C Illumina is limited to 300 bp read lengths and cannot be used for clinical diagnostics
• D Illumina performs massively parallel sequencing of millions of clonally amplified DNA clusters simultaneously in a flow cell ✓

Explanation

Illumina NGS generates billions of short reads simultaneously by sequencing millions of clonal DNA clusters in a flow cell in parallel, using fluorescently labelled reversible terminator dNTPs (cleavable 3'-O-azidomethyl blocking group). After each base incorporation, image is captured then the terminator is chemically cleaved to allow the next cycle. Sanger sequencing is capillary-based, reading one or at most 96 fragments per run, with longer reads (~1000 bp) but vastly lower throughput. Illumina's massively parallel nature enables whole-genome sequencing at clinical scale.

Reference: Harper's Illustrated Biochemistry, 32nd ed.

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Written and medically reviewed by the StethoPrep medical team.