Guide to Choosing a Sequencing Technique

When it comes to sequencing DNA, three main techniques available today offer excellent results—especially if you choose wisely. These methods—Sanger sequencing, next-generation sequencing (NGS), and long read sequencing—are sufficiently different that it behooves the novice sequencer to learn which one would be most appropriate for their research. Here is a brief review of these methods, advantages and disadvantages, and important criteria to help you weigh your next sequencing choice.

Criteria for choosing a sequencing method

Criteria for choosing a sequencing method are many, and depend on the researcher and the nature of their project. The biological question under study is key. “The context of the research determines what type of sequencing should best be used, as different sequencing methods have different characteristics that will drive selection,” says Jonas Korlach, Vice President and Chief Scientific Officer at Pacific Biosciences.

The most common criteria include sequencing time, throughput, cost, and accuracy—all of which tend to influence each other. “The dynamics between accuracy, cost, and data throughput have historically been the drivers of sequencing technology selection,” says Alex Aravanis, Chief Technology Officer, Head of Research and Product Development at Illumina. “Improvements in cost and throughput without sacrificing accuracy have driven the accessibility of DNA sequencing to a broad range of applications and users.”

Cost will depend on whether purchasing instrumentation is required, or already available to you (in the lab, department, or core facility); the purchase of materials and reagents; the number of samples; and whether you plan to outsource any sequencing or prep steps. Another factor worth considering is the size of the target(s) to be sequenced (i.e., a whole genome, or only a few genes), as this will affect sequencing time and cost.

As a consideration, sequencing time includes the overall time expected for project completion, but also the hands-on versus automated time. In addition, consider the throughput level of the sequencing technique, which affects the overall time expected for a project.

The reads lengths generated during the sequencing process may also factor into a decision, as longer read lengths are desirable for some applications (see below). NGS produces the shortest read lengths (about 50–500 bp), followed by Sanger sequencing (about 500–1000 bp), and then long read sequencing (about 5–30 kb). “Read length is a consideration to the extent that customers will sequence to a length required to answer a biological question, but not longer as it adds cost and time,” says Aravanis.

Sanger sequencing

Sanger sequencing is a time-tested technique that is straightforward and quick for a small number of samples, capable of resolving single base pairs. It works by chain termination upon incorporating a radiolabeled nucleotide analogue. The resulting DNA fragments are then separated by agarose gel electrophoresis for identification. The Sanger method is helpful for complex regions of DNA that contain repeats and secondary structures, and is often relied upon as an orthogonal method to verify genetic variants detected by NGS.

However, Sanger sequencing has a lower throughput compared to NGS, so it may not be appropriate for larger projects. “Sanger sequencing provides highly accurate sequencing of smaller DNA regions, a small set of genes, and typically fewer than 1000 samples,” says Jeffrey Smith, Global Lead of NGS Precision Medicine Initiatives at Thermo Fisher Scientific.

In fact, for projects of a smaller scope, using Sanger sequencing often makes the most sense. “The project time, cost, and complexity are much lower using Sanger sequencing for targeted or focused sequencing,” says Kay Eron, VP and General Manager of Capillary Electrophoresis at Thermo Fisher Scientific. Capillary electrophoresis uses Sanger sequencing chemistry within capillary tubes on a capillary electrophoresis instrument, and can be helpful for pinpointing individual bases in a gene fragment.

Next-generation sequencing

The size of the genetic target will strongly influence the choice of sequencing method. “While smaller DNA size and a small number of genes would be more appropriate for Sanger sequencing, a large area of genome, or panels of genes and gene targets, is best suited for NGS,” says Eron. Also known as massively parallel sequencing, NGS employs a sequencing-by-synthesis method using fluorescently tagged nucleotides in a massively parallel approach.

The greater throughput of NGS makes larger projects quicker, easier, and cheaper compared to using Sanger sequencing. “The project time and cost will be lower for NGS when sequencing large areas such as a genome or panel of genes,” says Eron. NGS is a good choice for whole genome sequencing, whole exome sequencing, analyzing large panels of genes, detecting rare variants, and discovery and diagnostics. “Discovery research scanning large areas of a genome for detections of new mutations, or sequencing a panel of genes all at once can be done efficiently with NGS technologies,” says Smith. “Sanger sequencing [for these applications] would simply take far too long and cost too much in both dollars and tissue.” The smaller amount of sample input required for NGS also makes it a good choice for analyzing rare samples.

The multi-discipline utility and scalability of NGS may be a decision point for some researchers. “NGS has extremely broad applicability across genomics, transcriptomics, and epigenetics with the ability to scale from small amplicon-based experiments to population-scale whole genome sequencing projects,” says Aravanis. However, for targeted sequencing (for example, of one or several genes), NGS can be more expensive than Sanger. NGS also requires access to the more expensive instrumentation of an NGS sequencer. The NGS workflow is more complex than that of Sanger sequencing, but the increasing use of automation is easing this burden.

Long read sequencing

In long read sequencing, DNA fragments are sequenced individually either as they move through a nanopore (Oxford Nanopore Technologies), or are copied within individual tiny wells (Pacific Biosciences). Longer overlapping sequences make assembly easier, like putting together a jigsaw puzzle with fewer, larger pieces, instead of the more numerous smaller pieces in NGS. Other advantages include that amplification bias is eliminated, and features such as large insertions/deletions, repetitive regions can be detected more easily. Long read sequencing can have a higher error rate than NGS, but ongoing technological improvements are closing the accuracy gap.

Long read sequencing is a good choice if shorter sequencing reads are not sufficient to answer your biological question. “Sequencing technologies that provide long read lengths (1 kb and longer) are helpful for genome assembly and detecting rare variants,” says Smith. “These applications lend themselves to this approach due to the need for more complete genomic coverage and a better understanding of the overall chromosome organization.”

Pacific Biosciences’ HiFi sequencing, using its SMRT technology, ensures high read accuracy for genomic studies that demand a comprehensive look at human genomes. “This reveals many more genomic regions, offers important phasing information, and provides full-length RNA transcript resolution, all of which are inaccessible to short-read sequencing,” says Korlach. “For example, in the area of rare and inherited disease research, researchers use PacBio HiFi sequencing for cases when short-reads are not sufficient and not providing answers.” PacBio is partnering with the UCLA Institute for Precision Medicine and David Geffen School of Medicine, and with Rady Children’s Institute for Genomic Medicine to help identify rare disease variants.

Sometimes a researcher might need to use two sequencing methods. “A small percent of the human genome is particularly difficult to read,” says Aravanis. “Nanopore and single molecule [long read sequencing] approaches could be used in a complementary manner to Illumina SBS [NGS], by acting as a reflex test or orthogonal validation, or in combination with Illumina SBS and other methods for de novo assembly and/or reference genome.” While this is unusual, it’s nice to know the possibility exists, and that advances in technology are making all methods increasingly convenient and affordable.