Sanger Sequencing vs Next Generation Sequencing: Key Differences

DNA sequencing has revolutionized genomics, allowing scientists to decode genetic information with increasing speed and accuracy. Two major technologies dominate this space: Sanger sequencing, developed in the 1970s, and next-generation sequencing (NGS), which emerged in the early 2000s. Each has distinct advantages depending on cost, throughput, and application.

Understanding their differences helps researchers select the right tool for tasks ranging from targeted gene analysis to whole-genome sequencing.

Chain-Termination Steps

Sanger sequencing relies on the incorporation of chain-terminating dideoxynucleotides (ddNTPs) during DNA synthesis, halting elongation at specific bases. DNA polymerase extends a growing DNA strand by adding nucleotides complementary to a template strand. When a ddNTP is incorporated instead of a regular deoxynucleotide (dNTP), the absence of a 3′-hydroxyl group prevents further extension. A mixture of dNTPs and fluorescently labeled ddNTPs generates DNA fragments of varying lengths, each terminating at a specific nucleotide.

The sequencing reaction occurs in a thermal cycler, where cycles of denaturation, annealing, and extension allow for progressive ddNTP incorporation. The resulting DNA fragments are separated by capillary electrophoresis, which sorts them by size. As fragments migrate through a polymer-filled capillary, a laser excites the fluorescent labels on the ddNTPs, and a detector records the emitted signals. This produces a chromatogram, where each peak corresponds to a nucleotide in the original sequence. The high accuracy of this method makes it ideal for clinical diagnostics and mutation validation.

NGS, in contrast, eliminates chain termination, using massively parallel sequencing-by-synthesis techniques. Instead of ddNTPs, NGS platforms use fluorescently labeled reversible terminators that temporarily halt elongation after each nucleotide addition. Optical detection captures the signal before the blocking group is removed, allowing further extension. This iterative process enables simultaneous sequencing of millions of DNA fragments, vastly increasing throughput.

Library Construction Techniques

Preparing DNA for sequencing involves transforming genetic material into a format suitable for sequencing platforms. This step, known as library construction, differs significantly between Sanger sequencing and NGS.

In Sanger sequencing, the process is straightforward, typically involving PCR amplification of a single DNA fragment or cloning into a plasmid vector. The resulting linear or circular DNA serves as a template for sequencing reactions. Since Sanger sequencing produces long reads—often 800 to 1,000 base pairs—minimal fragmentation is needed. The focus is on obtaining high-purity templates to avoid ambiguous chromatogram signals.

NGS requires a more complex library preparation process due to its high-throughput capabilities. Instead of sequencing a single DNA fragment, NGS platforms process millions of short DNA molecules simultaneously, necessitating extensive fragmentation and adapter ligation. DNA is first sheared into small fragments, typically 100 to 500 base pairs, using enzymatic digestion or mechanical methods like sonication. Adapter sequences are then ligated to the fragments, allowing hybridization to the sequencing flow cell and primer binding during sequencing.

Adapters also enable barcoding, where unique index sequences are added to different samples, allowing multiple samples to be pooled in a single sequencing run. This multiplexing capability reduces sequencing costs and increases efficiency. After adapter ligation, libraries undergo size selection to ensure uniform fragment lengths, which is critical for optimal cluster generation during sequencing. This step is performed using gel electrophoresis, bead-based purification, or automated systems. PCR amplification is often used to enrich for properly ligated fragments, though PCR-free protocols minimize amplification biases.

Reaction Platforms And Detection Strategies

Sanger sequencing and NGS operate on distinct reaction platforms. Sanger sequencing uses capillary electrophoresis-based instruments, where DNA fragments are separated by size and detected via fluorescence-based signal capture. This method, relying on single or multiple capillaries, limits the number of DNA molecules analyzed simultaneously. While highly accurate, its low throughput makes it less suitable for large-scale genomic studies. Instruments such as the Applied Biosystems 3730xl DNA Analyzer remain widely used for applications requiring high-fidelity sequencing, such as clinical validation and forensic analysis.

NGS platforms, in contrast, use massive parallelization to sequence millions of DNA fragments simultaneously. The most widely adopted NGS technology, Illumina sequencing, employs sequencing-by-synthesis on a solid-phase flow cell. DNA templates attach to the surface, where bridge amplification generates clusters of identical molecules. Each nucleotide incorporation is monitored in real time via fluorescently labeled reversible terminators, with high-resolution imaging capturing the emitted signals. This cycle of nucleotide addition, fluorescence detection, and signal processing enables rapid sequencing of entire genomes.

Other NGS platforms, such as the Ion Torrent system, use semiconductor-based detection, where hydrogen ions released during nucleotide incorporation generate an electrical signal, bypassing fluorescence imaging.

Third-generation sequencing technologies, such as Pacific Biosciences’ single-molecule real-time (SMRT) sequencing and Oxford Nanopore’s nanopore sequencing, further expand detection strategies. SMRT sequencing captures fluorescence from DNA polymerase molecules as they incorporate bases, allowing for ultra-long reads useful for resolving structural variants and repetitive regions. Nanopore sequencing measures electrical current changes as single DNA strands pass through a biological nanopore, providing real-time sequencing without amplification or chemical labeling. This portability makes nanopore sequencing valuable for field applications, such as infectious disease surveillance.

Throughput And Data Output

The volume of sequencing data generated by Sanger sequencing and NGS differs dramatically. Sanger sequencing has relatively low throughput, as each reaction processes a single DNA fragment at a time. While it delivers long, high-accuracy reads—often exceeding 800 base pairs—its capacity is limited by the need for individual capillary electrophoresis runs. This makes it impractical for large-scale genomic studies but well-suited for validating mutations or sequencing single genes. A typical automated Sanger sequencing run can process hundreds of samples per day, but the total number of bases sequenced remains far lower than what NGS can achieve.

NGS platforms generate massive amounts of data due to their ability to sequence millions of DNA fragments in parallel. Illumina’s high-throughput instruments, such as the NovaSeq 6000, can produce terabases of sequencing data in a single run, enabling whole-genome sequencing at population scales. The trade-off for this immense data output is shorter read lengths—often 100 to 300 base pairs—necessitating advanced bioinformatics tools for sequence assembly. Despite this, NGS has transformed fields like cancer genomics and microbiome research, where deep sequencing coverage is essential for detecting rare variants and complex microbial populations.