Transposase-Based Sequencing: Principle, Steps, Methods, Uses

 

Table of Contents

• Introduction to Transposase-Based Sequencing
• What are Transposases?
• Principle of Transposase-Based Sequencing
• Transposase-based Sequencing Methods 
• Steps of Transposase-Based Sequencing
• Advantages of Transposase-Based Sequencing
• Limitations of Transposase-Based Sequencing
• Applications of Transposase-Based Sequencing
• References

Introduction to Transposase-Based Sequencing

• Transposase-based sequencing uses enzymes called transposases for library preparation, enabling simultaneous DNA fragmentation and insertion of sequencing adapters.
• This method simplifies and speeds up the library preparation process.
• It allows the addition of barcodes, enabling multiple samples to be sequenced in a single run.
• It is applied in areas such as epigenetics, chromatin accessibility studies, structural variation analysis, and 3D genome mapping.
• Despite advancements in sequencing technology that have increased throughput and reduced costs, traditional library preparation methods have not evolved as rapidly.
• Conventional methods involve multiple steps: DNA fragmentation, end-repair, and adapter ligation.
• These traditional steps are time-consuming, require large amounts of DNA input, and can lead to sample loss.
• Transposase-based methods overcome these limitations by reducing preparation time, requiring less input DNA, and increasing overall throughput.

What are Transposases?

• Transposases are enzymes that catalyze the cutting and pasting of specific DNA sequences known as transposons or jumping genes, enabling these sequences to move within the genome through transposition.
• They naturally insert DNA randomly into a genome, a property utilized in sequencing to fragment DNA and simultaneously insert sequencing adapters in a process called tagmentation.
• The most widely used transposase in sequencing is Tn5 transposase.
• In sequencing workflows, Tn5 performs DNA fragmentation and adapter ligation in a single step.
• This single-step method replaces traditional multi-step workflows involving fragmentation, end-repair, A-tailing, and adapter ligation.
• After tagmentation, the resulting DNA fragments are amplified and sequenced.
• Many commercially available library preparation kits use transposase-based methods.
• Two popular examples of such kits are Nextera and seqWell.

Principle of Transposase-Based Sequencing

• Transposase-based sequencing, also called tagmentation-based sequencing, operates on the principle of tagmentation.
• It uses transposase enzymes to streamline and enhance the library preparation step by combining DNA fragmentation and adapter ligation into a single reaction.
• The transposase enzyme has a unique ability to cut DNA at specific sites and simultaneously insert adapter sequences into the resulting fragments.
• These adapters include specific recognition sites or barcodes that are essential for downstream sequencing steps.
• During the process, the transposase binds to the DNA and inserts adapter sequences at both ends of the DNA fragments.
• These adapter sequences are crucial for subsequent amplification and sequencing.
• This approach reduces the number of steps required for library preparation, making the sequencing process more efficient, faster, and cost-effective.

Transposase-based Sequencing Methods 

• ATAC-seq (Assay for Transposase-Accessible Chromatin Sequencing) is used to identify accessible chromatin regions by inserting sequencing adapters into open chromatin.
• These open regions are associated with active gene expression and are useful for studying gene regulation.
• It is widely applied in epigenomic and single-cell research.
• The Tn5 transposase cleaves DNA and inserts adapters into less condensed chromatin, which are then amplified and sequenced to identify active genomic regions.
• CUT&Tag (Cleavage Under Targets and Tagmentation) is a chromatin profiling technique used to study protein-DNA interactions.
• It detects specific chromatin regions associated with histone modifications or transcription factors.
• A specific antibody targets DNA-associated proteins and is linked to a Tn5 transposase.
• The transposase cleaves and inserts adapters at the protein-binding sites, and the tagged fragments are sequenced to reveal protein locations on the genome.
• Dip-C (Diploid Chromatin Conformation Capture) is a single-cell sequencing method used to map the 3D genome structure of individual diploid cells.
• It combines chromatin interaction capture methods like Hi-C with transposase-based tagmentation.
• The process includes chromatin fixation, digestion, and proximity ligation of interacting fragments.
• Tagmentation with Tn5 fragments the DNA and adds adapters; sequencing data is analyzed to understand chromatin folding patterns.
• Tn5mC-seq is used to study DNA methylation patterns by combining transposase-based tagmentation with bisulfite conversion.
• It uses a modified Tn5 transposase to tag methylated DNA regions and detects epigenetic modifications.
• Amplified and sequenced regions reveal precise locations of 5-methylcytosine (5mC).
• Unlike traditional bisulfite sequencing that needs large DNA inputs due to degradation, Tn5mC-seq uses very small samples and simplifies library preparation.
• LIANTI (Linear Amplification via Transposon Insertion) is an advanced single-cell whole genome amplification (WGA) method.
• It enhances accuracy in detecting mutations and structural variations.
• LIANTI uses linear amplification instead of traditional exponential methods, reducing amplification bias and errors.
• Tn5 transposase tags DNA at the single-cell level, enabling more accurate genomic DNA amplification.

Steps of Transposase-Based Sequencing

1. Sample Preparation

DNA is isolated from the biological sample, which may involve extracting genomic DNA or chromatin depending on the specific method. A transposase-adapter complex is then formed by combining Tn5 transposase with specific adapter sequences.

2. Tagmentation or Transposase Insertion

Transposase enzymes are added to the sample to fragment the DNA and insert adapters simultaneously. The enzyme cuts DNA at specific positions and inserts adapter sequences into the cut sites. These adapters contain sequences necessary for PCR amplification and may include barcodes for sample identification. This step is called tagmentation.

3. Purification

After tagmentation, excess transposase and any residual reaction components are removed through purification. This step ensures that only the tagged DNA fragments proceed to the next stages.

4. PCR Amplification

The adapter-tagged DNA fragments are amplified by PCR. The adapters facilitate amplification, ensuring sufficient quantities of each fragment are produced for sequencing.

5. Sequencing

The amplified DNA is sequenced using high-throughput sequencing platforms. The sequencing machine reads the DNA fragments and generates raw data.

6. Data Analysis

The sequencing data undergoes quality control, including removal of low-quality reads and adapter sequences. Reads are aligned to a reference genome and analyzed using bioinformatics tools to extract meaningful biological insights.

Advantages of Transposase-Based Sequencing

• Transposase-based sequencing simplifies the library preparation process, making it faster and more efficient than traditional methods.
• It reduces both the cost and time required for library preparation.
• It requires lower amounts of input DNA, making it ideal for low-input samples and single-cell applications.
• The method minimizes sample loss and enhances the overall efficiency of sequencing.
• By randomly inserting adapters into DNA, it enables the simultaneous processing of many DNA fragments.
• This random insertion increases sequencing throughput and supports large-scale studies.
• The process is simple and less technically demanding, making it accessible for a wide range of users.
• It is adaptable to various sequencing applications, including whole genome sequencing and targeted sequencing.

Limitations of Transposase-Based Sequencing

• Tn5 transposase performs best with longer DNA fragments, making it unsuitable for samples containing short DNA, which can lead to insertion bias and reduced adapter incorporation.
• Adapter contamination is a limitation; if adapters are not properly removed during data analysis, they can cause sequencing errors or mismatches.
• Commercially available transposase-based kits can be expensive, posing challenges for large-scale studies and specialized sequencing applications.
• Insertion site bias can result in uneven genome coverage, affecting the quality and consistency of sequencing data.
• Sample impurities or contaminants can interfere with transposase activity, lowering the efficiency and quality of sequencing.
• PCR amplification during library preparation may introduce amplification biases, which can compromise the accuracy and representativeness of sequencing results.

Applications of Transposase-Based Sequencing

• Transposase-based sequencing is used to study 3D genome structures, with methods like Dip-C employing transposase to create sequencing libraries and capture DNA interactions.
• Dip-C uses multiple barcodes, enabling high-resolution single-cell 3D genome structure analysis, surpassing the resolution of bulk Hi-C methods.
• It is valuable for detecting genomic variations, especially in single-cell genomics where precise variation detection is critical.
• In chromatin accessibility studies, ATAC-seq utilizes transposase to identify open chromatin regions linked to active gene expression.
• Tn5 transposase is applied in long-fragment sequencing (LFR), which is beneficial for de novo genome assembly and structural variation detection.
• Transposase-based methods are used to study epigenetic mechanisms such as histone modifications and DNA methylation.
• Techniques like Tn5mC-seq capture DNA methylation patterns with minimal DNA input, offering advantages over traditional whole-genome bisulfite sequencing (WGBS).
• The method also supports single-cell RNA sequencing, enabling rapid and efficient processing of individual RNA samples for transcriptomic analysis.

References

• Adey, A. C. (2021). Single-cell genomics using tagmentation-based methods. Genome Research, 31(10), 1693–1705. https://doi.org/10.1101/gr.275223.121
• Adey, A., & Shendure, J. (2012). Whole-genome bisulfite sequencing with ultra-low DNA input using tagmentation. Genome Research, 22(6), 1139–1143. https://doi.org/10.1101/gr.136242.111
• Chen, C., Xing, D., Tan, L., Li, H., Zhou, G., Huang, L., & Xie, X. S. (2017). LIANTI: Single-cell whole-genome analysis via linear amplification and transposon insertion. Science, 356(6334), 189–194. https://doi.org/10.1126/science.aak9787
• Fu, Z., Jiang, S., Sun, Y., Zheng, S., Zong, L., & Li, P. (2023). CUT&Tag as an advanced tool for profiling chromatin and epigenetic features. Epigenetics, 19(1). https://doi.org/10.1080/15592294.2023.2293411
• Kia, A., Gloeckner, C., Osothprarop, T., Gormley, N., Bomati, E., Stephenson, M., Goryshin, I., & He, M. M. (2017). Enhancing genome sequencing using an engineered transposase system. BMC Biotechnology, 17(1). https://doi.org/10.1186/s12896-016-0326-1
• Li, N., Jin, K., Bai, Y., Fu, H., Liu, L., & Liu, B. (2020). Applications of Tn5 transposase in modern genomics. International Journal of Molecular Sciences, 21(21), 8329. https://doi.org/10.3390/ijms21218329
• Picelli, S., Björklund, Å. K., Reinius, B., Sagasser, S., Winberg, G., & Sandberg, R. (2014). Tn5 transposase and scalable tagmentation workflows for high-throughput sequencing. Genome Research, 24(12), 2033–2040. https://doi.org/10.1101/gr.177881.114
• seqWell, Inc. (2025, February 26). NGS library preparation using transposase technology. Retrieved from https://seqwell.com/technology/
• seqWell. (2023, June 12). Advancing sequencing applications with enhanced transposase-based methods. Retrieved from https://seqwell.com/the-transcription-series-enabling-sequencing-applications-with-improved-transposase-based-solutions/