Tagmentation is a molecular biology technique that streamlines the initial steps of preparing a DNA sample for next-generation sequencing. It simplifies the lengthy process of converting large DNA molecules into a library of small, adapter-flanked fragments ready for sequencing. The term “tagmentation” is a portmanteau, describing a single-tube reaction that simultaneously fragments the target DNA and tags the resulting pieces with sequencing adapters. This consolidation of two traditionally separate steps—mechanical fragmentation and enzymatic adapter ligation—significantly accelerates the preparation workflow.
The Molecular Mechanism
The efficiency of tagmentation stems from its ability to perform both DNA cutting and molecular tagging in one enzymatic reaction. When the target DNA is introduced to the reaction mix, a specialized enzyme complex randomly accesses the double-stranded molecule at various points along its length. At each point of contact, the enzyme cleaves the DNA backbone, breaking the long molecule into smaller fragments.
Simultaneously with the cleavage, the enzyme inserts a short, synthetic DNA sequence—the sequencing adapter—directly into the newly created break site. This process results in a staggered cut, where the adapter sequence is covalently attached to the 5’ end of the DNA fragment on one strand, leaving a small, nine-nucleotide gap on the opposite strand. The reaction is completed in a single tube, meaning the final DNA fragments are already “tagged” for subsequent amplification and sequencing.
The Transposome Complex
The chemical engine that drives the tagmentation process is the transposome complex. This complex is a pre-assembled unit, consisting of a genetically modified transposase enzyme bound to the sequencing adapter DNA. The most commonly used enzyme is a hyperactive variant of the Tn5 transposase, derived from a naturally occurring bacterium.
The Tn5 transposase is naturally a “cut-and-paste” enzyme that facilitates the movement of genetic material within an organism’s genome. Scientists engineered this enzyme to remove its natural cargo and load it with the synthetic sequencing adapter sequences instead. The resulting transposome is stable and acts as a molecular scalpel, inserting the adapter sequences precisely at the site of DNA cleavage. The adapter sequence contains a 19-base pair segment called the Mosaic End, which the transposase specifically recognizes and uses to anchor itself before performing the reaction.
Efficiency and Input Requirements
A key benefit of tagmentation is the reduction in hands-on time and the overall duration of library preparation. Traditional methods involve hours of manual steps, including mechanical shearing and multiple enzymatic reactions for end-repair and adapter ligation, each requiring separate purification. Tagmentation replaces this multi-step protocol with a single, short enzymatic incubation, reducing the total assay time to less than two hours.
This streamlined workflow also requires less starting material than older techniques. Tagmentation protocols are sensitive and can successfully generate sequencing libraries from low input amounts, sometimes less than one nanogram of DNA. The ability to work with minute quantities is useful when dealing with precious or limited samples, such as those collected from clinical biopsies or forensic evidence. Furthermore, eliminating the need for expensive mechanical shearing devices and reducing reagents lowers the overall cost of library preparation.
Diverse Applications in Genomic Research
The technical advantages of tagmentation have made it a widely adopted technique across several fields of genomic research. The ability to process minimal amounts of DNA is beneficial for single-cell sequencing, where the entire genetic material from an individual cell may only be a few picograms. Tagmentation allows researchers to analyze the genome or transcriptome of thousands of single cells in parallel, providing high resolution into cellular heterogeneity.
Tagmentation is also a standard approach in metagenomics, which involves analyzing the complex mixtures of DNA found in environmental samples like soil or the human gut microbiome. The technique’s tolerance for diverse DNA sources and its ability to tag small, degraded fragments make it ideal for capturing the genetic diversity of a microbial community. Chromatin profiling techniques, such as the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), rely on the Tn5 transposome to fragment and tag only accessible regions of the genome, providing insight into gene regulation.