How do I choose the right sequencing technology?

As discussed in the article What are the different types of sequencing technologies?, there are two main types of sequencing technologies in use today: Sanger sequencing and next-generation sequencing (NGS). Sanger sequencing is based on a chain-termination method that utilizes radiolabeled, chemically modified analogues of the four nucleotide bases (A, T, G, and C). The analogues are termed dideoxynucleotides (ddNTPs) because they are missing the hydroxyl group that is required for extension of the chain. The chain terminates at A, T, G, or C once the analogue is incorporated, and the sequence is resolved by capillary electrophoresis (CE). Sanger sequencing technology uses capillary electrophoresis to generate sequencing reads >500 nucleotides in length. In contrast, NGS is a massively parallel sequencing method capable of generating hundreds of millions of shorter reads simultaneously. Both Sanger sequencing and NGS have a fast turnaround time, and both have utility in the field of genetic analysis.

The choice of sequencing technology depends on the research questions and goals of the project—which will help define the throughput, read length, and accuracy requirements—and the sample types available. Note that the choice is not mutually exclusive; because Sanger sequencing by CE remains the gold standard for base-calling applications where accuracy is critical, it is often used as an orthogonal method to verify variants detected by NGS. In the study of human genetic diseases, for example, the technology is chosen based on the level of phenotypic heterogeneity observed: Sanger sequencing may be chosen to generate reads up to 1,000 bp and analyze one or two genes in a disease with a clearly defined phenotype, or to confirm NGS variants. NGS may be chosen to discover novel variants in diseases with higher levels of phenotypic heterogeneity by sequencing larger numbers of genes. This is because NGS enables the interrogation of hundreds to thousands of genes at one time in multiple samples, as well as discovery and analysis of different types of genomic features in a single sequencing run, from single nucleotide variants (SNVs), to copy number and structural variants, and even RNA fusions.

Learn more about how scientists use sequencing technologies to answer challenging questions ›

Use the image and table below to help you determine which technology is best suited for your research needs.

Workflow steps for Sanger sequencing (top) and NGS (bottom).

* Automated library preparation of any 2-pool Ion AmpliSeq panel on the Ion Chef System.

To find out more about NGS, see the NGS Basics articles.

A note about fragment analysis

Capillary electrophoresis instruments are capable of performing both Sanger sequencing and fragment analysis; fragment analysis comprises a series of techniques in which DNA fragments are fluorescently labeled, separated by CE, and sized by comparison to an internal standard. While DNA sequencing by CE is used to determine the specific base sequence of a particular fragment or gene segment, this technique can also provide sizing, relative quantitation, and genotyping information for fluorescently labeled DNA fragments produced by PCR using primers designed for a specific DNA target.

Analysis of DNA fragments enables a multitude of applications, including cell line authentication, determination of CRISPR-Cas9 editing efficiency, microsatellite marker analysis, the multiplex ligation-dependent probe amplification (MLPA) assay, relative fluorescent quantitation or quantitative fluorescence PCR (QF-PCR), SNP genotyping, detection of aneuploidy, and more. While sequencing technologies also enable these applications, researchers choose fragment analysis because it has a faster turnaround time, higher sensitivity and is higher resolution. It is also more cost-effective due to its multiplexing capability; alleles for overlapping loci are distinguished by labeling locus-specific primers with different colored dyes. Due to this ability, fragment analysis enables the interrogation of more than 20 loci in a single reaction.

To find out more about fragment analysis, see the article What is fragment analysis?

Summary

Sanger sequencing, NGS, and fragment analysis each enable a variety of applications across a broad range of research areas. However, depending on the requirements of the project, one method may be chosen over another. Sanger sequencing is ideal for small-scale projects focusing on one or two genes, while NGS is ideal for higher-throughput sequencing needs. While fragment analysis does not provide sequence information, it can be used to generate relative quantitation information in a cost-effective manner due to its multiplexing capability.