Liquid biopsy stands at the forefront of diagnostic innovation, offering minimally invasive methods of analyzing DNA, proteins, and other molecules in bodily fluids as biomarkers of health and disease. This approach not only facilitates earlier disease detection and treatment monitoring but also offers insights into disease prognosis and progression with minimal patient discomfort, enhancing overall patient care.

Among the various liquid biopsy targets, circulating DNA stands out as particularly dynamic and impactful due to its adaptability and expanding use cases in cancer, prenatal testing, pathogen infections and beyond. Despite recent advancements in next-generation sequencing (NGS) technologies, which have significantly increased the accuracy, cost-effectiveness, and throughput of DNA analysis methods, these cell-free DNA (cfDNA)-based liquid biopsies still face significant challenges in mainstream clinical testing. Overcoming limitations such as low sensitivity and specificity, data management and interpretation, and the lack of standardized protocols will be essential to witness the integration of cfDNA liquid biopsies in routine disease screening and clinical testing.

Leading the charge in addressing these challenges, Wasatch Biolabs offers proprietary nanopore technologies that tackle some of the most significant obstacles in genetic and epigenetic testing. This article focuses on NGS-based liquid biopsies due to their expanding use cases and relevance in clinical testing. We discuss the role of NGS in liquid biopsies, technological advancements driving the global NGS market and the current movement towards , future clinical outlooks, and limitations.

High-throughput NGS technologies have revolutionized genomics and epigenomics, making it possible to rapidly and comprehensively analyze genetic material. In the context of liquid biopsy, NGS technologies have enhanced our ability to detect and characterize cfDNA, circulating tumor cells (CTCs), and other nucleic acid biomarkers with unprecedented accuracy and insights. With these advancements, NGS assays are increasingly accessible and affordable, setting the stage for widespread clinical diagnostics and healthcare implementation in oncology and beyond.

While cancer diagnostics have traditionally involved invasive tissue biopsies, which can be painful, risky, and potentially miss the full genetic picture of tumors, NGS-based liquid biopsies offer a simpler alternative: a blood draw to reveal key genetic mutations in circulating tumor-derived DNA (ctDNA) and, more recently, epigenetic modifications. Identifying these genomic features allows clinicians to tailor treatment plans to the patient’s unique genetic profile, optimizing outcomes and minimizing side effects. Because cfDNA is widely investigated in cancers and various other disease states, technologies assessing cfDNA will be highlighted in this article.

The past decade has witnessed significant improvements in the accuracy, throughput, and cost-effectiveness of NGS. Short-read platforms that read DNA in short 100-200 bp fragments like Illumina's NovaSeq have dominated the field and become essential tools in research and clinical settings. Offering comprehensive, whole genome technologies for broad use cases like biomarker discovery and rare and inherited disease diagnostics, and targeted sequencing technologies for higher-throughput clinical applications like population genetics, these technologies are breaking into other therapeutic areas outside of cancer.

In recent years, significant advancements have included increased read lengths for resolving difficult-to-sequence regions of the genome, enhanced accuracy and throughput, reduced costs, and improved data quality. Other advancements like ultra-deep sequencing allow the detection of low-frequency mutations in cfDNA despite noise and biases introduced by short-read sequencing processes¹. This capability is particularly important for liquid biopsy, where the total concentration of cfDNA can be very low and the concentration of cfDNA molecules of interest is even lower.

Additionally, new bioinformatics tools have been developed to enhance data analysis and interpretation. These tools filter out background noise to distinguish true genetic alterations from sequencing artifacts. Machine learning algorithms are also being more widely employed to predict treatment response and disease progression based on cfDNA profiles, further enhancing the clinical utility of liquid biopsies.

In 2023, the global next-generation sequencing market was valued at USD 9.19 billion and is projected to reach USD 66.04 billion by 2033, growing at a compound annual growth rate of 21.8%, according to a report by Nova One Advisor. This substantial growth highlights the increasing demand and adoption of NGS technologies across various fields, including clinical diagnostics and personalized medicine. The ability to rapidly generate large volumes of sequencing data, coupled with decreasing costs, is driving the expansion of the NGS market​​.

The COVID-19 pandemic further accelerated the adoption of NGS, with pharmaceutical and biotechnology companies leveraging these technologies to develop test kits and vaccines. For instance, researchers utilized Oxford Nanopore Technologies' affordable MinION Mk1C for sequencing SARS-CoV-2 (COVID-19) samples and tracking the evolution of new variants². This demonstrates the versatility and importance of NGS in responding to global health crises beyond oncology​.

The integration of NGS technologies into liquid biopsy has had a profound impact on clinical diagnostics and patient care. One of the most significant benefits is the ability to detect cancer at earlier stages. Early detection enables timely intervention and treatment, significantly improving survival rates³.

NGS-based liquid biopsies are also proving valuable for monitoring patient treatment responses. By analyzing cfDNA at multiple time points, clinicians can assess how well a patient is responding to a therapy and make necessary adjustments. This real-time monitoring is especially important for targeted therapies like cancer immunotherapies, where treatment efficacy can significantly vary among patients​.

Furthermore, liquid biopsy can provide insights into the mechanisms of drug resistance. By sequencing cfDNA from patients who develop resistance to treatment, researchers can identify genetic and epigenetic alterations that drive drug resistance and develop strategies to overcome them. This knowledge is critical for designing next-generation therapies that target resistant cancer cells more effectively​.

Liquid biopsy is most commonly integrated into cancer diagnostics, followed by prenatal testing, infectious disease detection, and treatment monitoring.

Despite the promising advancements, several challenges remain in implementing NGS-based liquid biopsies in routine clinical care. These challenges include demonstrating the diagnostic and economic benefits of NGS, managing complex and lengthy library preparation and target enrichment workflows, handling the large volumes of data produced by NGS, and standardizing bioinformatics analysis for more accessible and consistent data interpretation.

A primary challenge in proving the diagnostic benefits of NGS liquid biopsy is improving the sensitivity and specificity of cfDNA assays. Detecting low-frequency variants amidst normal DNA can be problematic, often resulting in false positives or negatives. Ongoing research is focused on developing more robust and accurate sequencing methods to address this issue. Fortunately, the automation of protocols and workflows is reducing the need for manual work, making processes more efficient and less prone to human error.

Data interpretation and standardization also pose significant challenges. The vast amount of data generated by NGS requires sophisticated bioinformatics tools for accurate analysis. However, the lack of standardized protocols and variability in data analysis methods can lead to inconsistent results. Establishing standardized guidelines and improving data interpretation algorithms are essential for ensuring the reliability of liquid biopsy tests at scale.

While the cost of sequencing has decreased significantly over the years, it remains a barrier to widespread adoption, especially in resource-limited settings. Reducing sequencing costs and improving accessibility will be critical for expanding the reach of liquid biopsy in clinical practice. Efforts to streamline and economize NGS processes are ongoing, aiming to make these advanced diagnostics available to a broader range of patients and healthcare providers⁴.

Enhancements in data generation and bioinformatics are crucial for improving sequencing data.  Automation and improved algorithms can address these challenges. Implementing standardized protocols will significantly boost data quality and accuracy across various fields.

NGS-based liquid biopsy technologies are revolutionizing clinical diagnostics and precision medicine. By providing accurate, comprehensive, and real-time genomic and epigenomic information, these technologies offer insights into personalized treatment strategies, early disease detection, and improved patient outcomes. As advancements continue, the integration of NGS into mainstream clinical testing will undoubtedly enhance the landscape of modern medicine.

This article serves as a foundation for understanding the advancements in NGS technologies and their applications in liquid biopsy. In upcoming articles, we will explore the transformative potential of rising long-read sequencing technologies and delve into the history and evolution of methylation analysis, setting the stage for the introduction of Wasatch BioLabs’ proprietary sequencing technologies.

Interested in learning more about Wasatch BioLabs’ leading NGS technologies? Visit www.wasatchbiolabs.com to explore our nanopore-based solutions and stay updated on the latest advancements in the field.

1. Gong B, Deveson IW, Mercer T, et al. Ultra-deep sequencing data from a liquid biopsy proficiency study demonstrating analytic validity. Scientific Data. 2022/04/13 2022;9(1):170. doi:10.1038/s41597-022-01276-8
2. Stüder F, Petit J-L, Engelen S, Mendoza-Parra MA. Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing. Scientific Reports. 2021/08/05 2021;11(1):15869. doi:10.1038/s41598-021-95563-w
3. Li W, Liu JB, Hou LK, et al. Liquid biopsy in lung cancer: significance in diagnostics, prediction, and treatment monitoring. Mol Cancer. Jan 20 2022;21(1):25. doi:10.1186/s12943-022-01505-z
4. Alexandrou G, Mantikas K-T, Allsopp R, et al. The evolution of affordable technologies in liquid biopsy diagnostics: the key to clinical implementation. Cancers. 2023;15(22):5434.