In principle, the concepts behind Sanger vs. next-generation sequencing (NGS) technologies are similar. In both NGS and Sanger sequencing (also known as dideoxy or capillary electrophoresis sequencing), DNA polymerase adds fluorescent nucleotides one by one onto a growing DNA template strand. Each incorporated nucleotide is identified by its fluorescent tag.
The critical difference between Sanger sequencing and NGS is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run. This high-throughput process translates into sequencing hundreds to thousands of genes at one time. NGS also offers greater discovery power to detect novel or rare variants with deep sequencing.
Advantages of NGS include:
“With Sanger sequencing, we saw a limited DNA snapshot… NGS and its massively parallel sequencing enable us to look at tens to hundreds of thousands of reads per sample.”
|Sanger Sequencing||Targeted NGS|
* Discovery power is the ability to identify novel variants.
† Mutation resolution is the size of the mutation identified. NGS can identify large chromosomal rearrangements down to single nucleotide variants.
‡ 10 ng DNA will produce ~1 kb with Sanger sequencing or ~300 kb with targeted resequencing (250 bp amplicon length × 1536 amplicons with TruSeq Custom Amplicon workflow)
NGS enabled Franco Taroni, MD to identify variants in a fraction of the time and at a significantly lower cost than Sanger sequencing.Read Interview
While Sanger sequencing was unable to detect rare variants, NGS could identify mosaicism down to a 1% limit of detection.Read Interview
Sanger sequencing offered a "limited DNA snapshot." Now, Michael Bunce, PhD uses NGS to look at hundreds of thousands of reads per sample.Read Interview
Sanger sequencing can be a good choice when interrogating a small region of DNA on a limited number of samples or genomic targets (~20 or fewer). Otherwise, targeted NGS is more likely to suit your needs. NGS allows you to screen more samples cost-effectively and detect multiple variants across targeted areas of the genome—an approach that would be costly and time-consuming using Sanger sequencing.
Find the right library prep kit for your sample type and application.
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With targeted resequencing, a subset of genes or a genomic region is isolated and sequenced, which can conserve lab resources. Learn more about targeted resequencing.
Whole-genome sequencing delivers a comprehensive view of genetic variation, ideal for discovery applications. Learn more about whole-genome sequencing.
Unlike Sanger sequencing, which analyzes only a single specimen at a time, NGS is effective for analyzing DNA from multiple species in large, complex environmental samples. Learn more about eDNA sequencing.