What are the genetic sequencing methods? Today's major technologies for gene sequencing are relatively mature, with the main methods for DNA sequencing being Sanger sequencing, next-generation sequencing (NGS) and long-read length sequencing. Below is a brief review of these methods, their advantages and disadvantages, and important criteria to help you weigh your next sequencing choice.

1. Criteria for Choosing a Sequencing Method

There are many criteria for selecting a sequencing method, depending on the nature of the researcher and their project. The biological question being studied is key, says Jonas Korlach, vice president of Pacific Biosciences, "The context of the study determines which type of sequencing is used, as different sequencing methods have different characteristics to drive the choice."

Common criteria include sequencing time, throughput, cost and accuracy - all of which tend to interact with each other. "The dynamic between accuracy, cost and data throughput has been a driving factor in the choice of sequencing technology, with cost and throughput improvements driving the adoption of DNA sequencing for a wide range of applications without sacrificing accuracy."

Sequencing lab costs include the cost of purchasing instruments, the cost of lab reagents and consumables, the number of samples, the planned outsourcing of sequencing and the pre-sequencing preparation steps. Another factor worth considering is the size of the target to be sequenced, which can also affect sequencing time and cost.

The read lengths generated during sequencing may also influence the decision, as some of these applications require longer read lengths. For example, NGS produces MIN read lengths (~50-500 bp), followed by Sanger sequencing (~500-1000 bp) and then long read length sequencing (~5-30 kb).

2. Sanger sequencing

Sanger sequencing is a tried and tested technology that is simple and fast for small numbers of samples and is capable of resolving individual base pairs. It works by strand termination after incorporation of radiolabeled nucleotide analogs. The Sanger method is useful for complex DNA regions containing repeats and secondary structures and is often used as an orthogonal method to verify genetic variants detected by NGS.

However, Sanger sequencing may not be suitable for large projects due to its lower throughput compared to NGS. Sanger sequencing allows for highly accurate sequencing of smaller DNA regions, a small set of genes, and typically less than 1000 samples.

In fact, for smaller scale projects, targeted or focused sequencing using Sanger gene sequencing methods is much less time, cost and complexity for projects that typically use fine tube electrophoresis, i.e.: Sanger sequencing chemistry within a capillary tube on a capillary electrophoresis instrument that helps to identify individual bases in a gene fragment.

3. Next generation sequencing

The choice of sequencing method can be heavily influenced by the sequencing target; Sanger sequencing is suitable for a small number of genes or short genomic fragments, while NGS sequencing is suitable for sequencing large genomes or whole genomes. Compared to Sanger sequencing, NGS sequencing has high throughput characteristics that allow large projects to be completed quickly, easily and cost-effectively. Therefore, NGS is more ideal for whole genome sequencing, whole exome sequencing, analysis of large genomes, detection of rare variants, and discovery and diagnosis. However, for targeted sequencing, NGS may be more expensive than Sanger. Also, the NGS sequencers used for NGS have higher instrumentation costs and the NGS workflow is more complex than Sanger sequencing.

4. Long-read long sequencing

In long read length sequencing, DNA fragments are sequenced individually as they pass through the nanopore (Oxford Nanopore Technologies) or are replicated in a single small well. The longer overlapping sequences make assembly easier, like putting together a puzzle with fewer large pieces rather than more small pieces in NGS. Benefits include elimination of amplification bias and easier detection of features such as large insertions/deletions, duplicated regions, etc. Long-read long sequencing may have a higher error rate than NGS, but continued technical improvements are closing the accuracy gap.