Overview of Tandem Repeats

What Are Tandem Repeats?

Tandem repeats are sequences of DNA comprised of two or more nucleotides that replicate consecutively along a chromosome in a continuous, head-to-tail arrangement. These repeating units, often referred to as repeat motifs, exhibit variability, spanning from a mere few repetitions to potentially hundreds within a specific chromosomal locale.

The Different Types of Tandem Repeats

Tandem repeats are distinguished by their diverse classification, encompassing micro forms, short and long variants, and microsatellites.

The two primary categories for tandem repeats are as follows:

• Short Tandem Repeats (STR) or Microsatellites

Microsatellites, also recognized as simple sequence repeats (SSRs), denote tandem repeats of DNA with motif sizes ranging from 1 to 6 base pairs (bp).

Analysis
• Variable Number of Tandem Repeats (VNTR)

Referred to interchangeably as minisatellites and microsatellites, VNTRs are tandem repeat units of DNA with a motif size of ≥7 bp. Although some literature designates motifs ≥100 bp as microsatellites, this usage lacks consistency and still classifies them as VNTRs under the defined criteria.

Crucially, the classification of tandem repeats is independent of the number of repetitions of the unit. Notably, a three-base-pair long STR (e.g., "ACG") repeated 10,000 times in tandem (totaling 30,000 base pairs) remains classified as an STR. Similarly, a motif of 50 bases repeated only three times (totaling 150 base pairs) would still be considered a VNTR, despite its shorter overall length compared to the first example. This classification underscores the emphasis on the size of the repeating unit and its individual units, irrespective of the total copy number.

The Origin of "Satellite DNA"

The origin of the term "satellite DNA" can be traced back to a time when DNA sequencing was not as advanced, accessible, or prevalent as it is today. Before the era of precise and widespread DNA sequencing, researchers employed alternative techniques to discern the composition of an organism's genome. The terminology, including "satellite DNA", "microsatellite" and "minisatellite", emerged from the initial characterization of specific genomic DNA segments during density gradient centrifugation.

In the mid-20th century, scientists utilized density gradient centrifugation to isolate DNA. During this process, they noted that genomic DNA exhibited distinct bands of varying densities. Some of these bands manifested as satellites, positioned apart from the primary genomic DNA band. Upon sequencing these DNA satellite bands, researchers discovered the presence of tandem repeats of different sizes, now collectively referred to as satellite DNA.

The distinction between tandem repeats of ≥7 bp being termed "variable number" tandem repeats (VNTRs) and smaller repeats being labeled as short tandem repeats (STRs) does not imply inherent variability in one over the other. The nomenclature is not indicative of a difference in mutability or variability between VNTRs and STRs, including the frequency of point mutations within their motifs or the variability in the number of repetitions in the genome. This classification is rooted in the size of the repeating unit, not the level of variability, emphasizing a historical and descriptive approach rather than a direct reflection of their dynamic characteristics.

Why It Is Important to Study the Mechanism of Tandem Repeats?

The significance of tandem repeats extends beyond their predominant presence in non-coding gene regions, playing a crucial role in biology with implications more profound than initially apparent. Comprising over 3% of the entire human genome, tandem repeats exert a substantial impact on structural genomic variation, particularly for sequences exceeding 50 base pairs. The marked variability within these tandem repeat regions underscores their pivotal role in shaping the phenotypes of numerous eukaryotic organisms.

Moreover, tandem repeats emerge as influential factors in the realm of human health. They have been identified as key players in the onset of various genetic diseases, elevating their importance in biomedical research. Tandem repeat sequences, when linked to changes in gene expression, have been implicated in numerous cancers and connected to more than 50 neurological disorders, such as ALS, FXS, ataxia, autism spectrum disorders, and schizophrenia. This highlights their relevance in understanding the molecular basis of diseases.

The identification, precise delineation, and cataloging of tandem repeat sequences represent the foundational steps in unraveling their disease-driving mechanisms. This intricate exploration holds the promise of unveiling potential biomarkers, elucidating drug targets, and fostering the development of therapeutics—an imperative pathway in advancing our understanding and treatment of diverse medical conditions.

The Features of Repeated sequence

In comparison to other genomic structures, repeated sequences exhibit distinctive characteristics that render them instrumental in various biological applications:

• Accelerated Evolutionary Rate and Species-Specific Markers

Repetitive sequences evolve at a faster pace, with certain sequences being species-specific. These species-specific repetitive elements serve as valuable genetic markers, facilitating the study of phylogenetic relationships among different species.

• Chromosome Fingerprinting and Karyotype Analysis

Repetitive sequences play a vital role in chromosome fingerprinting and karyotype analysis. This aids in the precise localization of exogenous chromosome segments. Additionally, SSR sequences derived from repetitive elements are employed in genetic map construction, gene localization, variety identification, and related applications.

• Detection and Identification of Exogenous Genetic Material

Repetitive sequences, particularly those scattered throughout the genome, serve as probes for the detection and identification of exogenous genetic material in different species.

• Epigenetic Modification and Gene Expression Regulation

Repetitive sequences contribute to epigenetic modifications, influencing the regulation of inserted or neighboring genes. This, in turn, modulates gene expression, impacting individual phenotypes, and influencing adaptability.

However, the intricate nature of the genome, coupled with the polymorphism of repetitive sequences and the challenges associated with assembly, complicates their identification. Despite these challenges, advancements in high-throughput sequencing technology, the decreasing costs of sequencing, and the continual development of sophisticated software algorithms are progressively overcoming these hurdles. As a result, the identification of repetitive sequences, especially those with high-frequency occurrences in the genome, is becoming more feasible and holds promise for enhanced genomic understanding.

Revolutionizing Tandem Repeat Research with Long-Read Sequencing and Bioinformatic Analysis

In contrast to antiquated density gradient centrifugation methods, contemporary scientists leverage advanced DNA sequencing technologies to unravel the intricacies of tandem repeats. Particularly, long-read sequencing platforms such as PacBio HiFi sequencing and Nanopore sequencing have become indispensable. These technologies, characterized by extended read lengths, empower researchers to precisely identify bases while seamlessly traversing extensive arrays of repetitive sequences with substantial overlap in read lengths.

The landscape of tandem repeat research has undergone a transformative shift, propelled by the integration of bioinformatic analysis tools tailored to complement PacBio HiFi sequencing. This innovative approach circumvents challenges associated with traditional methods, offering researchers extensive reads surpassing 10,000 base pairs, boasting high accuracy levels (99.9%), and featuring a suite of specialized analysis tools adept at navigating the complexities of tandem repeat investigations.

Key applications facilitated by this advanced approach include:

• Size Genotyping and Mosaic Estimation

Accurate determination of tandem repeat sizes and estimation of mosaic patterns within genomic sequences.

• Sequence Composition Analysis

In-depth analysis of sequence compositions, including identification of breaks and regions harboring multiple repeats.

• 5mC CpG Methylation Detection

Precise identification and characterization of CpG methylation at the 5mC level.

• Haplotype-Resolved Read Stacking and Visualization of Methylation Status

Comprehensive exploration of haplotype-resolved read stacking, coupled with visualization tools for discerning methylation status.

The synergy between long-read sequencing and biological data analysis marks a departure from past mathematical challenges. Researchers now possess the means to address pivotal questions regarding the role of these crucial genomic regions across a spectrum of genetic phenomena, spanning trait evolution to the intricate biology of inherited diseases.