Tn5 Transposase Applied in Genomics Research

Abstract

The development of high-throughput sequencing (next-generation sequencing technology (NGS)) and the continuous increase in experimental throughput require the upstream sample processing steps of NGS to be as simple as possible to improve the efficiency of the entire NGS process. The transposition system has fast "cut and paste" and "copy and paste" functions, and has been innovatively applied to the NGS field. For example, the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) uses high-throughput sequencing to detect chromatin regions accessible by Tn5 transposase. Linear Amplification via Transposon Insertion (LIANTI) uses Tn5 transposase for linear amplification, haploid typing, and structural variation detection. Not only is it efficient and simple, it effectively shortens the time for NGS sample library construction, realizes large-scale and rapid sequencing, improves sequencing resolution, and can be flexibly modified for more technological innovation.

Keywords: 3D genome structures; Tn5; epigenetics; genomic variation; long fragments; open chromatin.

Figure 1

Tn5 transposon structure and transposition mechanism. (A). Tn5 transposon structure consisting of a core sequence that encodes three antibiotics and two inverted IS50 sequences. The outside ends (OEs) bind to Tn5 transposases. (B). Scheme of the Tn5 transposition mechanism [11]. (C). Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. Tn5mC-seq is used in research studies on DNA methylation. Mate-pair applied to the amplification of long DNA fragments. Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) is applied to open chromatin (scale bar 1:600). Linear Amplification via Transposon Insertion (LIANTI) is applied to genomic variation. Dip-C is used in reconstructing the 3D structure of the genome.

Figure 2

Tn5 in reconstructing 3D genome structures. (A). Tn5 is used to form a composite with barcodes, and then adapters are added by PCR. (B). The traditional method of Tn5 transposition using two barcodes results in the loss of 50% of the genomic sequence information. (C). Dip-C sequencing library construction utilizes 20 barcodes, reducing the initial information loss to 1/20 after PCR [17]. (D). Tan et al. used Dip-C to simulate the 3D genome structure of a human cell [17].

Figure 3

Tn5 is used in the study single-cell genomic variations. (A). Exponential amplification results in bias and errors. The replication yields of DNA fragments A and B are 100% and 70%, respectively. For a final copy number of approximately 10,000, the final ratio of fragments A/B for exponential amplification is 8:1 [22]. (B). Linear amplification significantly reduces bias and errors. The replication yields of DNA fragments A and B are 100% and 70%, respectively. For a final copy number of approximately 10,000, the final ratio of fragments A/B for linear amplification is 1:0.7 [22]. (C). The scheme of LIANTI sequencing library construction [22].

Figure 4

Tn5 is used to study open chromatin. (A). The principles and processes of DNase-seq. (B). The principles and processes of FAIRE-seq. (C). The principles and processes of ATAC-seq. (D). The principles and processes of ATAC-Seq. (E). Comparison of multiple methods for studying open chromatin.

Figure 5

Tn5 is used in long-fragment sequencing. (A). PAGE analysis of transposase continuity. Tn5 transposons are used to target 1-kb PCR amplicons. Lane 1, treatment of transposome with SDS to remove transposase; Lane 2, transposome without SDS treatment is used as control. Lane 3, input DNA; Lane 4, a DNA marker. Tn5 transposase remains bound to DNA after transposition, and the protein-DNA complex dissociates only after the addition of the protein denaturant, SDS [30]. (B). Principle and scheme for LFR. (1) Physical separation of 100-130 pg of high-molecular weight DNA into 384 different wells. (2) Through several steps, all in the same well, without intermediate purification, genomic DNA is amplified, fragmented, and ligated onto a unique barcode adapter (3) that is merged to all 384 wells, purified, and introduced into Complete Genomics’ sequencing platform 10, (4) custom alignment program to map paired reads to the genome, and the barcode sequence is used to group tags into haplotypes. (5) The final result is the diploid genome sequence. (C) Schematic diagram of stLFR technology. This technique starts from the extracted long DNA and inserts the transposon sequence into the long DNA randomly. The DNA double-strand complementation principle is used to combine the product with a magnetic bead carrier with multiple copies of molecular tags. After two adapters, PCR amplification is performed, and library construction is finally completed.

Figure 6

Using Tn5 in epigenetics. (A). Principle and s cheme for Tn5mC. Tn5 transposases loaded with a methylated adaptor (brown) attack genomic DNA. Oligonucleotide replacement methods anneal the second methylated adaptor (purple) and perform gap repair. Bisulfite treatment converts unmethylated cytosine to uracil (gray) and PCR is performed to add primers (pink, green) that are compatible with the external flow cell. Methylation is represented as a black lollipop. (B). Principle and scheme of ATAC-me. The experimental procedure is similar to Tm5C, but it is only for methylation of open chromatin. (C). A new technology called the combinatorial indexing design (CoBATCH). Protein A is fused to the N-terminus of a Tn5 transposase to form Protein A-Tn5 (PAT). First, antibodies are used to target a specific protein that binds with chromatin DNA. Then, PAT transposomes are used to insert the adaptors into antibody-immunoprecipitated chromatin and the resulting DNA is sequenced. (D). Principle and scheme of a new chip-seq technology called itChIP. Cross-link samples (cells or tissues) are treated with SDS at 62 °C to loosen whole-genome chromosomes without affecting the binding of proteins to DNA. Using this treatment, Tn5 can evenly cut chromosomes without creating a preference for open regions. Finally, antibodies are used to pull the specific proteins that bind to chromosomal DNA with adapters that are ready for PCR amplification and sequencing.