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

Transposase-based sequencing is a method that uses enzymes called transposase for library preparation that can fragment DNA and insert specific sequencing adapters into DNA fragments at the same time.

This makes the library preparation process faster and simpler. It also adds barcodes which allows multiple samples to be sequenced at once. It has applications in epigenetics, chromatin accessibility studies, structural variation analysis, and 3D genome mapping.

In recent years, sequencing technology has advanced significantly which has improved the sequencing throughput while reducing costs of sequencing. However, the process of library preparation has not kept up with these advancements. Traditional library preparation methods follow the same basic multi-step process which includes DNA fragmentation, end-repair, and adapter ligation. This process is time-consuming, requires large DNA input, and can result in sample loss. Transposase-based methods address these challenges by reducing preparation time, using less input DNA, and increasing throughput.

What are Transposases?

Transposases are enzymes that catalyze the cutting and pasting of specific DNA sequences called transposon or jumping genes and allow these sequences to move from one location to another through the process of transposition.

• It has the natural ability to insert DNA randomly into a genome. This ability has been used in sequencing to fragment DNA and simultaneously add sequencing adapters in a process called tagmentation.
• The most widely used transposase in sequencing is Tn5 transposase. In sequencing, Tn5 combines the process of DNA fragmentation and adapter ligation in a single step. 
• This method replaces traditional multi-step workflows that involve fragmentation, end-repair, A-tailing, and adapter ligation with a single-step reaction. The DNA fragments are then amplified and sequenced.
• Many commercially available library preparation kits use transposase. Two popular examples are Nextera and seqWell.

Principle of Transposase-Based Sequencing

Transposase-based sequencing, also known as tagmentation-based sequencing, works on the principle of tagmentation. It uses transposase enzymes which simplifies and improves the library preparation step of sequencing by combining the fragmentation and adapter ligation steps in a single reaction. The enzyme transposase has the unique ability to cut DNA at specific locations and simultaneously insert adapter sequences into the DNA fragments. The adapters contain specific recognition sites or barcodes which are important for later steps in sequencing.

In transposase-based sequencing, the transposase enzyme binds to the DNA and inserts adapter sequences at both ends of the DNA fragments. These adapters are needed for amplification and sequencing. This reduces the number of steps in the library preparation process and makes 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 regions are associated with active gene expression and are hence useful for understanding gene regulation. It is widely used in epigenomic and single-cell studies. The Tn5 transposase cleaves DNA and inserts sequencing adapters into less condensed regions. These regions are then amplified and sequenced to determine which areas of the genome are open and active.

• CUT&Tag (Cleavage Under Targets and Tagmentation) is a chromatin profiling method that is used to study protein-DNA interactions. It can identify specific chromatin regions where histone modifications or transcription factors are located. In this method, a specific antibody is used to target proteins that are associated with DNA. The antibody is linked to a Tn5 transposase which then cleaves and inserts adapters at the protein-binding sites. The tagged fragments are sequenced to identify where the proteins are located 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 by combining chromatin interaction capture methods like Hi-C with transposase-based tagmentation. The process begins with chromatin fixation and digestion followed by proximity ligation to join DNA fragments that are close. Then tagmentation is done which uses transposases like Tn5 to fragment DNA and add sequencing adapters. The DNA is sequenced and the sequencing data is analyzed to identify chromatin folding patterns.
• Tn5mC-seq is a method that uses Tn5 transposase to study DNA methylation patterns. This method combines transposase-based tagmentation with bisulfite conversion. It uses a modified Tn5 transposase to tag methylated regions of DNA and provides information about epigenetic modification across the genome. These regions are amplified and sequenced to identify the precise locations of 5-methylcytosine (5mC) modifications. Traditional bisulfite sequencing requires large amounts of DNA due to degradation during bisulfite treatment. On the other hand, Tn5mC-seq uses very few samples and makes library preparation easier and faster.
• LIANTI (Linear Amplification via Transposon Insertion) is an advanced method for single-cell whole genome amplification (WGA). This method improves accuracy in detecting mutation and structural variations. Unlike traditional amplification methods, this method uses linear amplification to reduce bias and errors. It uses Tn5 transposase to tag DNA at the single-cell level which leads to a more accurate amplification of genomic DNA.

Steps of Transposase-Based Sequencing

1. Sample Preparation

The first step is isolating DNA from the biological sample of interest. This may include extracting genomic DNA or chromatin from the sample depending on the method. Then, the transposase-adapter complex is formed by combining Tn5 transposase and specific adapter sequences.

2. Tagmentation or Transposase Insertion

In the next step, transposase enzymes are added to the sample which insert adapters into these DNA fragments. The transposase cuts the DNA at specific positions and simultaneously inserts the adapter sequences into the cut sites. The adapters contain sequences for PCR amplification and also may contain sample-specific barcodes. This process is called tagmentation.

3. Purification

After the tagmentation step, excess transposase or residual reaction components are removed by a purification step. This ensures that only the DNA fragments that have been tagged by the transposase remain for the next steps. 

4. PCR Amplification

The tagged DNA is amplified using PCR. The adapters allow the amplification of the fragmented DNA. This amplification step ensures that enough copies of each tagged fragment are generated for sequencing.

5. Sequencing

Finally, the amplified DNA is sequenced using high-throughput sequencing platforms. The sequencing machine reads the DNA fragments and the generated data is processed and analyzed.

6. Data Analysis

The raw sequencing data undergoes quality control including removal of low-quality bases and adapter sequences. Then, the reads are aligned to a reference genome. The aligned reads are analyzed using different bioinformatics tools to obtain relevant biological information.

Advantages of Transposase-Based Sequencing

• Transposase-based sequencing simplifies the library preparation process. This method can lower costs and reduce time compared to traditional library preparation methods. 
• It has lower DNA input requirements which makes it suitable for low-input samples and single-cell applications.
• It also reduces sample loss and improves the efficiency of sequencing.
• Transposase-based sequencing involves randomly inserting adapters which allows for simultaneous processing of many DNA fragments. This increases the sequencing throughput and allows large-scale studies.
• The process is also simple and less technically demanding which makes it useful for various applications. It can also be adapted for different types of sequencing applications including whole genome sequencing and targeted sequencing.

Limitations of Transposase-Based Sequencing

• Tn5 transposase works best with longer DNA fragments so it is unsuitable for samples with short DNA. This can cause bias and reduce the insertion of adapters.
• Another limitation is adapter contamination. If the adapters are not removed properly during analysis, they can cause errors or mismatches during data analysis. 
• Commercial transposase-based kits can be expensive. This can be a problem for large-scale studies and specialized sequencing methods.
• Insertion site bias is also a limitation that can lead to uneven genome coverage.
• Impurities or contaminants in the sample can also affect the efficiency of the transposase enzyme and lead to reduced quality of the sequencing data.
• The process of PCR amplification can introduce amplification biases which can reduce the accuracy of sequencing.

Applications of Transposase-Based Sequencing

• Transposase-based sequencing can be used to study 3D genome structures. Newer chromatin conformation capture methods Dip-C uses transposase to create sequencing libraries and can capture DNA interactions. Dip-C uses multiple barcodes which is useful in single-cell 3D genome structure analysis. This achieves higher resolution than bulk Hi-C methods.
• It is also useful in detecting genomic variations, especially in single-cell genomics. 
• It can be used in chromatin accessibility studies to identify open chromatin regions by using ATAC-seq.  
• Tn5 is also used in long-fragment sequencing (LFR). This is useful for de novo assembly and structural variation detection.
• It is also used to study epigenetic mechanisms like histone modifications and DNA methylation. Methods like Tn5mC-seq can capture methylation patterns with minimal DNA input compared to traditional whole-genome bisulfite sequencing (WGBS).
• It also has applications in single-cell RNA sequencing to rapidly and efficiently process single-cell RNA samples.

References

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