Next Generation Sequencing: A Review

https://meditropics.com/next-generation-sequencing-a-review/

*Dhruv Bansal, ** Abhishek Sharma, **Prashant Aggarwal

*Post Graduate resident, **Senior Resident,

Department Of Medicine, Lady Hardinge Medical College, New Delhi

 

Introduction

James Watson and Francis Crick discovered the double helical structure of the DNA in 1953 (1). This seminal event marked one of the most important milestones in the history of science and gave rise to field of modern molecular biology. However, it took mankind another twenty-five years before the first generation of DNA sequencing techniques, known as Sanger sequencing (2), was developed . This traditional method uses a “sequencing by synthesis” approach to determine nucleic acid sequences one nucleotide at a time. However, it was a slow, expensive and time consuming process. Even after the process was completely automated by Applied Biosystems in 1987, it took researchers more than $2.7 billion and over ten years to sequence the human genome, as part of the human genome project (3). To address the limitations of the Sanger technique, new methods collectively known as Next Generation Sequencing (NGS) were developed.

NGS has revolutionized the field of genomics by enabling rapid and cost-effective analysis of DNA and RNA sequences. This technology has far-reaching implications in various fields, from basic research to clinical applications. In this review, we delve into the clinical implications of NGS, its challenges, and future directions.

 

1.      A Brief Overview of Next Generation Sequencing

NGS is a powerful technology that allows for the rapid and high-throughput sequencing of DNA or RNA. Whereas Sanger sequencing is only capable of producing a sequence for one template per reaction, the NGS is typified by performing millions to billions of individual sequencing reactions simultaneously in a process referred to as massively parallel sequencing. The approach has revolutionized DNA sequencing to the point that a whole human genome can now be sequenced within 3 days. (3).

The process involves several steps, each crucial for obtaining accurate and reliable sequence data. The general steps involved are as follows –

1.1) Library Preparation:

Library preparation refers to the preparation of nucleic acid templates (DNA or RNA) for sequencing. First, the DNA or RNA is extracted from the sample of interest, whether it’s from cells, tissues, blood, or other sources. Next, it is fragmented into smaller pieces which can be done mechanically (such as sonication) or enzymatically (using restriction enzymes for DNA). Following this step, short DNA sequences called adapters are ligated to the ends of the fragmented DNA or RNA molecules. These adapters contain sequences that are complementary to those used in subsequent steps of the sequencing process.

1.2) Bridge Amplification:

The DNA fragments with adapters are then attached to a solid surface (like a flow cell) through the adapter sequences. During bridge amplification, the fragments are amplified into clusters, each containing thousands of identical copies of the same DNA fragment. This allows for simultaneous sequencing of many fragments, ensuring high-throughput sequencing, where multiple DNA fragments can be sequenced in parallel, accelerating the sequencing process.

1.3) Sequencing:

The clusters on the flow cell are now ready for sequencing. Single-stranded DNA molecules from the clusters are then denatured and a sequencing primer is hybridized to the template DNA. The sequencing instrument cycles through four steps for each nucleotide:

  1. A) Nucleotide Incorporation: One of the four nucleotides (A, C, G, or T) labelled with a fluorescent tag is added to the flow cell.
  2. B) Detection: A camera takes images to identify which nucleotide was incorporated in each cluster.
  3. C) Fluorescent Group Removal: The fluorescent tag is chemically removed to allow the next nucleotide to be added in the next cycle.
  4. D) Signal Readout: The signal from the incorporated nucleotide is recorded.

These steps are repeated hundreds or thousands of times to generate millions of sequences in parallel. The result is a massive amount of short sequence reads that cover the entire genome or transcriptome.

1.4) Data Processing, Analysis and Interpretation:

The raw data from the sequencing instrument, which consists of images and intensity signals, is converted into nucleotide sequences through a process called base calling. The resulting sequences (reads) are aligned and mapped to a reference genome or transcriptome. This step helps identify where each read originated from in the genome or transcriptome. The final step involves interpreting the data, which may include identifying genetic variations associated with diseases, studying gene expression levels etc.

2.      Clinical Applications
2.1) Precision Medicine

NGS has paved the way for personalized medicine, allowing for a deeper understanding of genetic variations and their associations with diseases. By sequencing an individual’s genome, clinicians can identify genetic predispositions to diseases, tailor treatments based on genetic markers, and predict drug responses. This approach has been particularly impactful in oncology, where targeted therapies are guided by the genetic profile of tumours. For example, NGS can identify specific mutations in cancer genes (e.g., EGFR, BRAF) to guide the selection of targeted therapies, improving patient outcomes. The Cancer Genome Atlas (TCGA) project is a seminal study that utilized NGS to profile genomic alterations in various cancer types. The project provided comprehensive genomic data, aiding in the identification of driver mutations and potential therapeutic targets.

2.2) Rare Disease Diagnosis

NGS has transformed the diagnosis of rare genetic disorders, which often have diverse and complex genetic causes. Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) allow for the comprehensive analysis of an individual’s genetic makeup, enabling the identification of rare variants responsible for diseases. This has led to faster and more accurate diagnoses for patients with rare diseases, potentially saving lives and improving quality of life. In a recent study by Yang et al (4), the authors conducted WES in 250 probands for whom referring physicians ordered WES. Using NGS, the authors were able to identify the causative genetic defect in 25% of patients who would otherwise have remained undiagnosed.

2.3) Prenatal Screening and Diagnosis

In prenatal care, NGS offers non-invasive prenatal testing (NIPT) for detecting chromosomal abnormalities such as Down syndrome, with higher accuracy than traditional methods. Moreover, NGS can analyse foetal DNA circulating in maternal blood, reducing the need for invasive procedures like amniocentesis. This has significantly improved prenatal screening and diagnosis, providing expecting parents with valuable genetic information about their unborn child.

2.4) Infectious Disease Genomics

NGS plays a crucial role in infectious disease genomics, facilitating the rapid and accurate identification of pathogens. By sequencing the genomes of infectious agents, such as bacteria and viruses, scientists can track outbreaks, study transmission patterns, and monitor the evolution of pathogens. The Ebola virus outbreak in West Africa in 2014 was a pivotal moment for NGS in infectious disease genomics. Quick et al. (5) utilized NGS to sequence the Ebola virus genomes, providing real-time data on viral evolution and aiding in outbreak control strategies. They were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 minutes. This pattern was repeated in the recent COVID19 outbreak as well. Soon after clusters of cases were initially identified in Wuhan, China, in December 2019, scientists began working on identifying the causative organism. The pathogen was identified as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the sequence was first reported to the local authorities on 5 January 2020 and was released on the open-access virology website virological.org on 11 January 2020 (6). This rapid molecular diagnosis aided in the rapid identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and was instrumental in the development of vaccines, saving millions of lives.

3.      A few Challenges

Next Generation Sequencing has undoubtedly revolutionized genomics and greatly impacted clinical practice, but it also comes with its own set of challenges. These challenges, if not addressed adequately, can hinder the full potential of NGS in clinical applications.

3.1) Data Analysis and Interpretation

One of the most significant challenges associated with NGS is the massive amount of data generated from sequencing runs. The sheer volume of information presents a computational challenge in terms of storage, processing, and analysing the data. NGS produces gigabytes to terabytes of raw sequencing data, and handling and interpreting this data requires robust bioinformatics pipelines and specialized skills. Bioinformatics analysis tools and algorithms play a critical role in transforming raw sequencing data into meaningful biological insights. Furthermore, the interpretation of NGS data to identify clinically relevant variants and mutations requires sophisticated algorithms and databases, adding another layer of complexity to the data analysis process.

 

3.2) Quality Control and Standardization

Ensuring the quality and reliability of NGS data is another challenge that needs to be addressed. NGS technologies can be sensitive to various sources of error, including sequencing errors, sample contamination, and technical artifacts introduced during library preparation and sequencing. Establishing standardized protocols for quality control and validation of NGS data is crucial to minimize variability and ensure the accuracy of results across different laboratories and platforms. Furthermore, the lack of standardized procedures for variant interpretation and reporting poses a challenge in the clinical implementation of NGS, especially in the context of personalized medicine and patient care.

3.3) Clinical Validation and Regulatory Considerations

Integrating NGS into clinical practice requires rigorous validation of the technology for diagnostic and prognostic applications. Clinical validation involves demonstrating the analytical and clinical validity of NGS assays, ensuring that the tests reliably detect and characterize genetic variants associated with specific diseases or conditions. Additionally, regulatory considerations, such as obtaining approval or clearance from regulatory bodies like the FDA, add another layer of complexity to the implementation of NGS in clinical settings. Navigating the regulatory landscape and compliance with evolving standards and guidelines for NGS-based tests is a multifaceted challenge that demands collaboration between researchers, clinicians, and regulatory authorities.

3.4) Ethical and Legal Implications

The widespread use of NGS in clinical practice also raises ethical and legal challenges related to privacy, consent, and data sharing. The generation of vast amounts of genomic and genetic information brings forward concerns about patient privacy, the potential misuse of genetic data, and the need for informed consent and counselling. Ensuring responsible and ethical use of NGS data in the context of patient care and research requires careful consideration of legal and ethical frameworks and the development of policies to safeguard patient rights and confidentiality.

3.5) Cost and Accessibility

While NGS technologies have significantly reduced the cost of genomic sequencing, the initial investment in equipment, reagents, and infrastructure can still be prohibitive for many clinical laboratories, particularly in resource-limited settings. Additionally, ongoing costs associated with data storage, analysis, and maintenance of NGS instruments can strain institutional budgets. Addressing the cost and accessibility of NGS technology is crucial for its widespread adoption in clinical practice and for ensuring equitable access to genomic testing across diverse patient populations.

To summarize, while NGS holds immense promise for advancing precision medicine and improving patient outcomes, addressing the aforementioned challenges is critical to realizing its full potential in clinical applications. Collaborative efforts in bioinformatics, quality control, validation, regulatory compliance, ethics, and cost optimization are essential to overcome these challenges and ensure the effective integration of NGS into routine clinical practice.

4.      Future of Genomics

As we look to the future of genomic research and sequencing, several promising developments and advancements are anticipated to further propel the field forward. One of the most significant areas of focus is the continued refinement and optimization of NGS technologies. These efforts aim to address the existing challenges associated with data analysis, interpretation, and standardization, ultimately enhancing the clinical utility and reliability of genomic sequencing in patient care.

Advancements in NGS platforms and bioinformatics tools will play a crucial role in streamlining data analysis and interpretation. Efforts to develop more efficient algorithms and software solutions for processing large-scale genomic data are underway, aiming to reduce computational burden and improve the accuracy and speed of variant identification and interpretation. Additionally, the integration of artificial intelligence and machine learning algorithms into NGS data analysis holds great promise in enhancing our ability to extract clinically relevant insights from complex genomic datasets.

Furthermore, the future of genomic research and sequencing is intrinsically linked to the ongoing pursuit of precision medicine. As we gain a deeper understanding of the complex interplay between genetics, environment, and disease, NGS technologies will continue to drive the development of personalized therapeutic approaches and targeted interventions. The identification of genetic biomarkers and actionable mutations through genomic sequencing will play a pivotal role in guiding treatment decisions, enabling the delivery of more precise and effective patient care.

In parallel, the future of genomic research will also witness the convergence of multi-omics data, integrating information from genomics, transcriptomics, proteomics, and metabolomics to provide a comprehensive molecular understanding of health and disease. This holistic approach to data integration and analysis has the potential to uncover novel disease mechanisms, identify predictive biomarkers, and facilitate the discovery of new therapeutic targets, ultimately shaping the future of precision healthcare.

Moreover, as the field of genomics continues to advance, efforts to address the ethical, legal, and social implications of genomic sequencing will remain a priority. Establishing robust frameworks for data privacy, consent, and responsible data sharing will be essential to maintain patient trust and uphold ethical standards in the use of genomic information for clinical and research purposes.

As we navigate the complexities and challenges inherent in genomic research, collaborative efforts across scientific, clinical, and regulatory domains will be instrumental in realizing the full potential of genomics for improving patient outcomes and advancing the frontiers of medicine.

5.      Conclusion

In conclusion, Next Generation Sequencing has revolutionized genomics and is reshaping clinical practice. Its applications in precision medicine, rare disease diagnosis, prenatal screening, infectious disease genomics, and more, highlight its immense potential. However, challenges such as data interpretation, cost, and standardization remain to be addressed. As technology advances and collaborations strengthen, NGS is poised to continue transforming healthcare, offering new insights into diseases and improving patient outcomes. Efforts to overcome challenges and harness the full potential of NGS will undoubtedly drive the future of medicine towards more personalized, effective, and tailored approaches to patient care.

 

References

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  2. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec; 74(12):5463–7.
  3. Kumar KR, Cowley MJ, Davis RL. Next-Generation Sequencing and Emerging Technologies. Semin Thromb Hemost. 2019 Oct; 45(07):661–73.
  4. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, et al. Clinical Whole-Exome Sequencing for the Diagnosis of Mendelian Disorders. Available from: https://www.nejm.org/doi/full/10.1056/NEJMoa1306555 [cited 2024 Mar 13].
  5. Quick J, Loman NJ, Duraffour S, Simpson JT, Severi E, Cowley L, et al. Real-time, portable genome sequencing for Ebola surveillance. Nature. 2016 Feb 11; 530(7589):228–32.
  6. Zhang YZ, Holmes EC. A Genomic Perspective on the Origin and Emergence of SARS-CoV-2. Cell. 2020 Apr 16; 181(2):223–7.