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The Latest in Sequencing Technologies

3d render of dna structure for transposon sequencing

How Transposons are Changing the Sequencing Industry

Introduction

Mobile DNA segments that can change position, transpose, are transposons (or transposable elements). These “jumping genes” discovered by Barbara McClintock 70 years ago exist in species of all kingdoms, from bacteria to humans. Transposons make up large portions of a species’ genome and have key biological functions. In humans, transposons make up nearly 50% of the genome; however, only a very small portion of the transposons are still actually able to transpose.

Transposons induce mutations, can change the size of the genome, regulate gene expression, and more. These abilities are associated with a number of biological functions. In bacteria, one function is related to bacterial antibiotic resistance. In humans, transposons, such as the Alu family of repetitive elements, are associated with a number of diseases including cancers and genetic disorders.

The mutagenic abilities of transposons have been harnessed for use in insertional mutagenesis (inducing mutations by inserting new genetic material into a gene). This technology has been used to identify genes associated with cancer development. The combination of transposon insertional mutagenesis with sequencing techniques is the basis of transposon sequencing.

Utility of Transposon Science for Sequencing

There are four types of transposon sequencing methods:

  1. Transposon sequencing (TnSeq)
  2. Insertion sequencing (INSeq)
  3. High-throughput insertion tracking by deep sequencing (HITS)
  4. Transposon-directed insertion site sequencing (TraDis)

The basic process common to all four methods involves cleaving pooled DNA from a transposon insertion library created using insertional mutagenesis. Adaptors are attached to the DNA fragments containing the transposon sequences, then massive parallel sequencing [i.e., next-generation sequencing (NGS)] is conducted to ascertain transposon location sites1.

The primary advantage of transposon sequencing methods is the ability to obtain valuable information regarding gene function. Due to the high-throughput feature of transposon sequencing using massive parallel sequencing, it is the method of choice when the goal is to study the genotype-phenotype interactions of pathogenic microorganisms on a significantly larger scale. The application of transposon sequencing has been used for Pseudomonas aeruginosa bacteria (a cause of various infections in humans) to identify genes for antibiotic resistance2. The technology has also been applied to identify important genes in Porphyromonas gingivalis, a pathogen responsible for certain periodontal diseases3.

Transposon sequencing technology has been applied to identify previously difficult-to-ascertain genetic players involved in cell transformation4. Using TnSeq, Iskow et al., found new transposon insertions (L1) that occur at high frequencines in human lung tumor genetic material. For example, an L1 insertion was detected in lung-tumor derived cells, but was absent from matched normal DNA taken from the blood of the same patient. This suggests that lung tumor genomes support extensive L1 mobilization. They further found that alterations in DNA methylation may be associated with the mobilization of these transposons5.

Lenka et al. used transposon technology to disrupt gene function in embryonic stem cells to identify genes and associated phenotypes involved in stem cell differentiation6. Further, research endeavors using transposon sequencing have revealed the critical roles for retrotransposons (transposons that exhibit reverse transcription similar to retroviruses) in neurogenerative diseases, alcoholism, and post-traumatic stress disorder7. By applying transposon sequencing technology, new knowledge has been gained regarding genes that have relevance for colorectal cancer progression. Using transgenic mice with loss of apc gene function, which leads to intestinal tumorigenesis, they found that those mice with mobilized transposons showed a much earlier onset of disease when compared to controls8.

Industry Benefits of Transposon-related Sequencing

The new sequencing technologies contribute to the accumulation of massive amounts of data in significantly shorter timeframes. Translating the massive information and data resulting from next-generation sequencing efforts requires new bioinformatic tools to analyze and glean actionable information. For example, Zomer et al. has developed a web interface and open source software tool to analyze and determine essential genes from TnSeq-derived data9.  Keane et al. has also developed a bioinformatics tool, RetroSeq, to accurately find non-reference transposon insertions using NGS-derived data10.

The impact of the technology on medical diagnosis and therapy is yet to be more fully realized. However, the gene function component gleaned from the ever-increasing collection of biomedical data is already providing knowledge that can be used to determine the association of sequence data to actual functional outcomes (such as the role of a mutation to potential disease states). Technological advances in the conduct of transposon sequencing supports the investigation of disease pathogenesis, bolstering the identification of mechanisms promoting disease (i.e., cancer promotion). This in turn can lead to the identification of more definitive diagnostic biomarkers of disease and the design of therapies targeting specific disease-promoting processes. The use of NGS with transposon mutagenesis helps in the accessibility of the technology, thereby enhancing discovery and future use at the medical practice level.

Conclusions

Understanding the mechanisms underlying the genetic basis of phenotypes, including disease phenotypes, provides new opportunities to characterize the relationship between genetic mutations/sequences and the consequences to the organism. The expanding sequencing industry offerings will help researchers to decipher the role of transposons in the genomic changes leading to human disease. The combination of transposon mutagenesis with massive parallel sequence is a powerful tool for high-throughput interrogation of a genome allowing major advances in the functional genomics research arena in timeframes not previously possible.

These state-of-the art sequencing tools are important for the development of better diagnostic capabilities and therapeutic approaches. The highly automated and large-scale nature of NGS also adds costs benefits achieved due to the lower costs associated with massive parallel sequencing technology. The increased availability of transposon sequencing opens many new doors to biomedical discovery and in the understanding of evolutionary and disease processes.

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Resources

  1. van Opijnen T, Camilli A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nature reviews Microbiology. 2013;11(7):435-42.
  1. Gallagher LA, Shendure J, Manoil C. Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq. mBio. 2011;2(1):e00315-10.
  1. Klein BA, Duncan MJ, Hu LT. Defining essential genes and identifying virulence factors of Porphyromonas gingivalis by massively parallel sequencing of transposon libraries (Tn-seq). Methods in molecular biology (Clifton, NJ). 2015;1279:25-43
  1. Ranzani M, Annunziato S, Adams DJ, Montini E. Cancer gene discovery: exploiting insertional mutagenesis. Molecular cancer research. 2013;11(10):1141-58.
  1. Iskow RC, McCabe MT, Mills RE, Torene S, Pittard WS, Neuwald AF, et al. Natural mutagenesis of human genomes by endogenous retrotransposons. Cell. 2010;141(7):1253-61.
  1. Lenka N, Krishnan S, Board P, Rangasamy D. Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation. Stem cell reviews. 2014;10(3):408-16.
  1. Reilly MT, Faulkner GJ, Dubnau J, Ponomarev I, Gage FH. The role of transposable elements in health and diseases of the central nervous system. The Journal of neuroscience. 2013;33(45):17577-86.
  1. March HN, Rust AG, Wright NA, ten Hoeve J, de Ridder J, Eldridge M, et al. Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis. Nature genetics. 2011;43(12):1202-9.
  1. Zomer A, Burghout P, Bootsma HJ, Hermans PW, van Hijum SA. ESSENTIALS: software for rapid analysis of high throughput transposon insertion sequencing data. PloS one. 2012;7(8):e43012.
  1. Keane TM, Wong K, Adams DJ. RetroSeq: transposable element discovery from next-generation sequencing data. Bioinformatics (Oxford, England). 2013;29(3):389-90.

 

Written by Macrogen Corp.

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