What is a key advantage of next‑generation sequencing compared with traditional Sanger sequencing?

Key Advantage of Next-Generation Sequencing Over Sanger Sequencing

The primary advantage of next-generation sequencing over traditional Sanger sequencing is the ability to sequence hundreds of thousands to millions of DNA fragments simultaneously in a massively parallel manner, rather than sequencing a single DNA fragment at a time. 1, 2

Fundamental Technical Difference

• Sanger sequencing can only process one DNA sequence per reaction, requiring separate reactions for each DNA fragment to be analyzed 1
• NGS platforms perform massively parallel sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules simultaneously in a flow cell 3
• This parallel processing capability allows NGS to sequence hundreds of thousands of sequences simultaneously without requiring a cloning step before sequencing 1
• Modern NGS platforms can generate sequence outputs ranging from hundreds of megabases to multiple terabases per run, compared to single reads from Sanger sequencing 1

Why the Other Options Are Incorrect

Cost Considerations

• NGS is not universally less expensive per sample when considering instrument costs and setup 1
• While NGS offers lower cost per base sequenced ($10-$400 per billion bases depending on platform), the instruments themselves are expensive ($1,000 to $850,000) 1
• For small-scale testing or single-gene analysis, Sanger sequencing may actually be more cost-effective 1

Technical Expertise Requirements

• NGS requires substantially MORE technical expertise, not less, compared to Sanger sequencing 4
• NGS demands dedicated bioinformatics infrastructure, high-performance computing clusters, and specialized databases that were not traditionally part of laboratory workflows 4
• The analytical pipeline requires expertise in primary analysis (base calling), secondary analysis (quality assessment, alignment), and tertiary analysis (biological interpretation) 4
• Many clinical laboratories currently lack routine bioinformatic expertise needed for NGS implementation 4

Accuracy Comparison

• NGS is not more accurate than Sanger sequencing 1
• Sanger sequencing has a detection limit of approximately 15-20% variant allele frequency and is considered the "gold standard" for mutation detection 1
• NGS platforms have estimated error rates of 0.01% to 15% depending on the technology, with some platforms prone to specific errors like homopolymer errors or GC bias 1
• Sanger sequencing is often used to confirm NGS findings precisely because of its superior accuracy for single-nucleotide variant detection 1, 5

Clinical Implications of Parallel Sequencing Capability

• The simultaneous sequencing capability allows comprehensive multi-gene panel testing from limited tissue samples, avoiding the tissue depletion that occurs with sequential single-gene Sanger sequencing 6
• For conditions with genetic heterogeneity (multiple possible causative genes), NGS can evaluate all relevant genes in a single run rather than requiring multiple sequential Sanger reactions 5, 6
• This parallel capacity enables detection of multiple mutation types (point mutations, insertions, deletions, fusions, copy number variants) across numerous genes simultaneously 1, 6