STSE_1_1_01-09

Introduction

Genome and transcriptome sequencing enable the diagnosis of genetic illnesses (Gorzynski et al., 2022; AlDubayan et al., 2020), facilitate research into gene-trait connections for pharmacological and agricultural applications, support reference genome development, provide genetic variant annotation resources, and inform imputation approaches (Manolio, 2010; Peloso et al., 2019; Snelling et al., 2020; Liao et al., 2023; Karczewski et al., 2020; Li et al., 2009). Primarily, attempts to evaluate sequencing accuracy relied on indirect criteria, such as the proportion of variant calls that were transitions rather than transversions, or whether the results were in accordance with Mendelian inheritance (Eberle et al., 2017). The genome revolutionised the capacity to evaluate precision in a Bottle standards, which comprise a collection of seven human cell lines that underwent thorough genome characterisation using various technologies, analytical methodologies, and human curation (Wagner et al., 2022). The capacity to identify gains in accuracy beyond what existing individual approaches can achieve was enhanced by this resource, which, when coupled with community competitions and comparisons, sparked a surge of innovation in sequencing and analysis (Olson et al., 2022; Foox et al., 2021; Wenger et al., 2019; Tetikol et al., 2022; Scheffler et al., 2023) techniques that prove their inventions are valid. Tests have demonstrated that this innovation can detect disease-causing variations that were previously undetected, thereby improving diagnosis rates. A new approach to sequencing, developed by Element Biosciences, utilises avidity sequencing instead of synthesis sequencing. This method is reportedly superior to Illumina sequencing in terms of per-base accuracy. It can yield high-yield short-read sequencing, with over six 30× genomes in a single sequencing run (AlDubayan et al., 2020).

The purpose of this study is to provide a concise overview of nuclear genomes in three-dimensional structure from a broad perspective, encompassing progress in DNA sequencing applications and the development of new generations of sequencing technology.

Materials and Methods

This review follows a narrative synthesis approach. Literature searches were conducted in PubMed, Web of Science, and Scopus databases between January 2000 and March 2025. Search terms included combinations of:

“Sanger sequencing”
“next-generation sequencing”
“third-generation sequencing”
“long-read sequencing”
“Hi-C”
“chromatin conformation capture”
“3D genome”
“structural variation”

Inclusion criteria comprised primary methodological studies, platform validation reports, benchmarking analyses, and authoritative review articles published in peer-reviewed journals. Non-English articles, conference abstracts without full text, and non-peer-reviewed sources were excluded. Selected studies were analyzed for technical principles, read length, error profiles, throughput, sequencing depth requirements, and application domains.

An overarching perspective on nuclear genomes in three-dimensional structure

In the nuclei of eukaryotic cells, the genetic code is stored. It is encased in chromatin, dispersed across several chromosomes, and folded into intricate three-dimensional structures with protein factors and RNA molecules. Traditionally, microscopy-based approaches were used to study nuclear architecture and chromatin spatial organization within the nuclear space (Lowenstein et al., 2004). Amphibian and other animal oocytes display the earliest known chromatin loop structure, the “lampbrush chromosome” (Morgan, 2002). However, early imaging methods lacked the resolution necessary to comprehensively characterize genome-wide chromatin interactions.

The development of high-throughput sequencing technologies enabled a paradigm shift in studying genome architecture. Genome-wide chromatin conformation capture techniques, particularly Hi-C and ChIA-PET, have become central tools for interrogating three-dimensional genome organization (Lieberman-Aiden et al., 2009; Fullwood et al., 2009). These approaches combine proximity ligation with massively parallel sequencing to generate contact maps representing interaction frequencies between genomic loci across the entire genome.

Hi-C enables unbiased, genome-wide detection of chromatin interactions by crosslinking spatially proximal DNA fragments, digesting them with restriction enzymes, ligating interacting fragments, and subjecting the resulting chimeric products to deep sequencing (Lieberman-Aiden et al., 2009). The resolution of Hi-C interaction maps is strongly dependent on sequencing depth, with higher read counts allowing finer-scale detection of chromatin loops, topologically associating domains (TADs), and chromatin compartments (Dixon et al., 2012). Achieving kilobase-scale resolution requires extensive sequencing coverage, often reaching billions of read pairs, thereby underscoring the essential role of next-generation sequencing platforms in 3D genome research.

ChIA-PET integrates chromatin immunoprecipitation (ChIP) with proximity ligation and deep sequencing to identify chromatin interactions mediated by specific DNA-binding proteins (Fullwood et al., 2009). This method allows high-confidence mapping of regulatory loops associated with transcription factors such as CTCF or RNA polymerase II (Tang et al., 2015). Compared to Hi-C, ChIA-PET provides protein-centric interaction maps but requires high-quality antibodies and substantial sequencing depth to achieve robust signal detection.

The hierarchical organization of the mammalian genome is now widely recognized, largely based on sequencing-driven discoveries over the past 15 years. Chromosome territories represent large-scale nuclear organization units, within which chromatin compartments segregate into transcriptionally active (A compartments) and inactive (B compartments) regions (Lieberman-Aiden et al., 2009; Ouyang et al., 2020). At a finer scale, TADs partition chromosomes into self-interacting domains (Dixon et al., 2012), while chromatin loops often anchored by CTCF constitute the fundamental structural units of 3D genome architecture (Tang et al., 2015).

Despite their transformative impact, sequencing-based chromatin conformation methods are subject to technical and analytical biases. Restriction enzyme selection, fragment length distribution, GC-content bias, PCR amplification efficiency, and ligation efficiency may influence interaction frequency measurements and coverage uniformity. Consequently, robust computational normalization methods are required to correct for systematic biases and ensure reproducibility of contact matrices (Lieberman-Aiden et al., 2009). Sequencing depth, library complexity, duplicate read rates, and normalization algorithms critically determine the resolution, reproducibility, and interpretability of 3D genome datasets.

Imaging-based approaches have further validated and complemented sequencing-based findings, strengthening our understanding of genome spatial organization (Wang et al., 2016; Yang and Wang, 2024). Due to space limitations, the present review focuses primarily on sequencing-based technologies and their contribution to 3D genome biology.

Chromosome territories at the macro level, compartments at the micro level, topologically associated domains (TADs), and fine-scale loops comprise the genome›s hierarchical structure acronym: CCCTC-binding factor, or CTCF for short. Several short-read sequencing methods were detailed by Kumar et al. (٢٠٢٤), including nanoball sequencing, ion semiconductor sequencing, and sequencing by synthesis. Bypassing the shortcomings of short-read sequencing, third-generation long-read sequencing is expected to resolve repeat sequences and massive genomic rearrangements with greater reliability. Advancements in complementary techniques and massively parallel DNA sequencing have enabled a deeper understanding of the biological underpinnings of disease causes. Nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing are just a few of the emerging approaches that will continue to drive the rapid evolution of this field. Precision and individualised medical treatment are the future promises of these cutting-edge technologies, which have several possible uses in the treatment of haematological illnesses (Kumar et al., ٢٠٢٤). It was discovered by Akintunde et al. (٢٠٢٥) that there is a desire to clarify the precise sequencing and content of DNA molecules.

Breakthroughs in decoding the DNA sequence and the subsequent procedures have made new opportunities in research, biotech, and healthcare possible. Humanity and the global economy have benefited from, and will continue to benefit from, the use of high-throughput sequencing technologies in these sectors. A few hundred base pairs can be sequenced in days thanks to advancements such as the use of fluorescent dyes and the polymerase chain reaction (PCR) for amplification. Automation, on the other hand, made it possible to sequence thousands of base pairs in hours. There has been significant progress, but it could be even better. In this article, we will examine the background and current state of next-generation sequencing systems, as well as their potential applications in fields beyond biomedical research (Akintunde et al., ٢٠٢٥). According to Chai and Ruan (٢٠٢٥), chromatin consists of nuclear proteins and RNA. Its dynamic structure affects nuclear and genomic processes. High-throughput DNA sequencing technologies, such as Hi-C and ChIA-PET, have enabled the genome-wide mapping of chromatin folding structures, contributing to the development of ٣D genome biology since ٢٠٠٩. This field has experienced rapid growth over the past ١٥ years of development. It›s improving our understanding of how genomic organisation influences nuclear functions in biological systems. In this study, we discuss the advances in sequencing-based technologies for mapping ٣D genomic landscapes and the future of ٣D genome biology (Chai and Ruan, ٢٠٢٥). According to Carroll et al. (٢٠٢٥), when comparing whole-genome sequencing of Element to Illumina sequencing at the same coverage, the former achieves better mapping and variant calling accuracy. However, at lower coverages (٢٠-٣٠x), there are bigger discrepancies. They are measuring the base error rates of element reads and have found that these regions, particularly those containing homopolymers and tandem repeats, have reduced error rates. As an alternative to the usual short-read sequencing, we leverage Element›s capability for paired-end sequencing with larger insert sizes. Long insert element sequencing provides more precise genome analysis across all coverages, and our results demonstrate that larger insert sizes lead to even greater accuracy (Carroll et al., ٢٠٢٥).

Progress in DNA sequencing›s practical uses

DNA sequencing has numerous applications in science, healthcare, and agriculture, and it has significantly transformed the field of genetics. The potential uses of DNA sequencing technology have increased significantly over the years due to technological advancements and the development of new applications. The use of DNA sequencing has made great strides in personalised medicine, among other areas. The process of DNA sequencing enables the detection of genetic variants and mutations that may be associated with certain health issues. Medical practitioners can create individualised treatment programs by analysing a patient›s genome. The cancer diagnostic and therapeutic fields have benefited greatly from this strategy, as genetic testing has enabled the identification of tailored treatments and the prediction of treatment response (Gargis et al., ٢٠١٦). The investigation of microbiomes is another field that has benefited from DNA sequencing. To maintain our health, microbiomes—communities of microbes that inhabit our bodies—are essential. The genetic composition of these microbes and their interactions with humans can be studied by DNA sequencing. Novel approaches to treating inflammatory bowel disease and infectious disorders have resulted from this (Dethlefsen et al., ٢٠٠٧). The agricultural sector is another field that has benefited from the application of DNA sequencing. Crops and cattle can benefit from genetic differences associated with desirable qualities, such as drought resilience or disease resistance, which can be discovered using DNA sequencing. New breeding strategies can be developed using this information to create crops and cattle with improved characteristics and better adaptation to certain conditions. As a result, environmental science found new uses for it. Soil and water microbe communities can be studied by DNA sequencing (Tanksley and McCouch, ١٩٩٧).

Different sequencing technology generations

Reading technology DNA sequences have undergone fast evolution in the last ٢٠ years (Sakamoto et al., ٢٠٢٠; Goto et al., ٢٠٢٠). This lightning-fast development has allowed for major advances in DNA sequencing, which in turn have given rise to three distinct generations of sequencing technology (Figure ٢).

There have been three distinct waves of advancement in sequencing technology throughout the last 40 years. Sanger sequencing, which laid the groundwork for DNA sequencing, represents the first generation of sequencing methods. Platforms like Illumina and Ion Torrent, which enabled massively parallel sequencing in the second generation, allowed for high-throughput sequencing. Among the present third-generation options, we find PacBio and Nanopore, which provide the ability to sequence individual molecules as well as long reads (Satam et al., 2023).

Technologies for first-generation sequencing

Initial efforts in DNA and RNA sequencing relied on chemical degradation or enzymatic cleavage to separate molecules into manageable pieces for individual analysis. In 1964, Robert Holley used ribonuclease from Saccharomyces cerevisiae to sequence Alanine tRNA, the first nucleic acid molecule to be sequenced (Holley et al., 1965). The chemical degradation method that enabled the sequencing of whole bacteriophage PhiX174 was similarly created by Walter Gilbert and Allan Maxam (Heather and Chain, 2016). Nonetheless, Fredrick Sanger’s creation of the chain termination-based sequencing approach was the true game-changer (Sanger et al., 1977). This method enabled the generation of sequence readings that were several hundred nucleotides long by utilising dideoxynucleotides, which terminate the chain elongation of DNA strands during replication. Because it enabled the rapid sequencing of DNA and RNA, Sanger’s approach was widely adopted and transformed molecular biology (Heather and Chain, 2016). Launched in 1987 in the US, the Applied Biosystems ABI 370 was the first commercial automated sequencing machine. Automating the Sanger sequencing process with fluorescently labelled dideoxynucleotides and capillary electrophoresis, this machine greatly improved the accuracy and speed of DNA sequencing (Schuster, 2008). The rapid adoption of the ABI 370 marked the beginning of a technological revolution that would eventually give rise to higher-throughput sequencers capable of generating longer reads. Although newer, higher-throughput sequencing technologies have largely replaced first-generation technologies, they remain a significant historical milestone in the evolution of sequencing methods. The capacity to sequence RNA and DNA has ushered in a new era of discovery and progress in the study of genetics and molecular biology, which, in turn, has transformed many branches of medicine and biology (Schuster, 2008; Hutchison, 2007).

Technologies for second-generation sequencing

Because second-generation sequencing technologies can sequence thousands to millions of DNA fragments simultaneously, they have revolutionised the DNA sequencing industry. When compared to conventional Sanger sequencing, these techniques stand out due to their parallel sequencing capabilities. One of the most popular second-generation sequencing platforms is Roche’s 454, which utilises pyrosequencing a technique that enables the identification of sequences by detecting the release of pyrophosphate when nucleotides are added to a DNA template to determine the DNA copies. As an additional tool, Ion Torrent sequencing may identify the DNA sequence by monitoring the discharge of hydrogen ions as the manufacturing process progresses. Using reversible dye terminators, the Illumina sequencing platform enables sequencing by synthesis. This technology is widely employed in the field. The sequencing method known as SOLiD sequencing uses reversible terminators in a ligation-based approach to find the DNA sequence. Thanks to these second-generation sequencing methods, DNA sequencing is now significantly faster and has increased throughput, opening up numerous new possibilities in genomics and medical diagnostics. These systems have enabled targeted sequencing, transcriptome analysis, and whole-genome sequencing, which have enhanced our understanding of heritable diseases and facilitated more personalised medical care (Pervez et al., 2022).

Technologies for third-generation sequencing

The most recent developments in DNA sequencing, known as third-generation sequencing technologies, offer novel methods that overcome the drawbacks of earlier generations. These innovations enable long-read sequencing, which, compared to older techniques, allows for the reading of substantially larger DNA fragments. For instance, PacBio Sequencing allows for long-read sequencing of DNA fragments as long as tens of kilobases in length by employing a single-molecule, real-time (SMRT) method with fluorescently labelled nucleotides. Oxford Nanopore sequencing is another method that utilises nanopore technology to detect DNA sequences. This method involves passing a single-stranded DNA molecule through a nanopore and measuring the changes in electrical current. The sequencing technology offered by Oxford Nanopore enables large read lengths, high mobility, and real-time analysis (Jain et al., 2016; Roberts et al., 2013).

Conclusion

Significant progress has been made in DNA sequencing in the last several years. Researchers and physicians now have easier and more inexpensive access to DNA sequencing as a result of the significant decrease in cost and time required for the process brought about by high-throughput sequencing technology. Researchers are facing new challenges in data analysis and interpretation due to the rapid increase in sequencing data resulting from the decline in sequencing costs. The development of increasingly precise DNA sequencing technologies has opened up a wealth of new possibilities in fields as diverse as infectious disease tracking, understanding the hereditary basis of traits and behaviours, and detecting mutations that cause disease. As a result, genetics, medicine, and biology have all made significant strides forward. In addition, DNA sequencing has numerous potential applications in fields such as environmental microbiology, personalised medicine, metagenomics, and many more. The further expansion of DNA sequencing and analysis of previously inaccessible genomic regions has been made possible by the development of new sequencing techniques, such as third-generation sequencing and nanopore sequencing.

Ackn owledgement

The authors thank the University of Mashreq, Baghdad, Iraq, for providing institutional support and access to academic resources.

Novelty Statement

This review provides a concise yet integrated synthesis of the evolutionary trajectory of DNA sequencing technologies—from first-generation Sanger sequencing to third-generation long-read platforms—with a specific focus on their enabling role in three-dimensional genome mapping. Unlike broader sequencing reviews, this work explicitly links technological capabilities (e.g., read length, throughput, error profiles) to methodological requirements of chromatin conformation capture techniques such as Hi-C and ChIA-PET. It highlights recent advances including avidity sequencing and emerging long-read applications, offering a timely perspective on how sequencing innovations continue to shape 3D genomics and precision medicine.

Author’s Contribution

Bahaa Burhanuldeen Kargule: Writing original draft. Zain Ulabdeen Naser Hussein: Literature search, data extraction. Qays Qays Qandeel and Fahad Bahaa Aljanabi: Review & editing

Generative AI and AI assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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