Next Generation Sequencing in Pathology: Revolutionizing Diagnosis and Treatment

Next-generation sequencing (NGS) is transforming cancer diagnosis and treatment by offering new ways to examine the genetic makeup of tumors. Unlike traditional methods, which are limited to analyzing a handful of genes, NGS can simultaneously analyze hundreds of genes and generate valuable information necessary for accurate diagnosis, personalized treatment, and improved patient outcomes.

Next-Generation Sequencing

Next-generation sequencing is having major impacts on pathology and cancer medicine. In contrast to traditional DNA sequencing methods, which analyze one molecule at a time, NGS makes it possible to sequence millions of DNA molecules simultaneously, offering a much faster and more comprehensive analysis.

How NGS Works

Next-generation sequencing (NGS) involves a series of steps, each contributing to accurate data generation.

Sample Preparation

  • DNA isolation: Techniques like silica-based column chromatography or magnetic bead-based methods are used to capture DNA molecules from the sample (e.g., blood, tissue). This helps minimize contamination from other components like proteins and RNA.
  • Quality control: Stringent measures are used to determine DNA purity and quantity using techniques like gel electrophoresis and spectrophotometry. High-quality DNA is important for proper library preparation and sequencing.
  • DNA quantitation: The exact amount of DNA is determined using techniques like fluorometry or quantitative PCR(qPCR) to make sure that there is enough material.

Library Preparation

  • Fragmentation: The purified DNA is enzymatically or mechanically sheared into smaller fragments with a defined size range that is needed for the efficient library construction and sequencing reactions that follow.
  • Adapter ligation: Short, double-stranded adapter sequences are attached to the ends of the fragmented DNA molecules. These adapters play crucial roles in the sequencing process by providing priming sites for enzyme binding and facilitating fragment immobilization.
  • Size selection: Techniques like gel electrophoresis or magnetic beads are used to select DNA fragments that are within a specific size range, which is necessary for uniformity in the library and optimal sequencing performance.


  • Cluster amplification: Adapter-ligated DNA fragments are amplified millions of times on a flow cell surface which creates clonal clusters (copies) of each unique fragment. This step is necessary to generate a strong enough signal for accurate determination of the DNA sequence (base calling) during the subsequent sequencing steps.
  • Sequencing by synthesis: This method determines the nucleotide sequence of each fragment in the amplified clusters. It involves incorporating fluorescently labeled nucleotides into the growing DNA strand. The specific emitted fluorescence signal from each incorporated nucleotide identifies the added base.

Data Analysis

  • Demultiplexing: After library preparation, bioinformatic tools use these barcodes to assign sequenced reads back to their originating samples, ensuring data integrity and preventing cross-contamination.
  • Read alignment: Sophisticated algorithms map individual reads (short DNA fragments) to the reference genome or transcriptome. This step considers sequence similarity, potential mismatches (single nucleotide variations), and insertions/deletions to accurately place each read within its rightful genomic context, which is crucial for variant calling and interpretation.
  • Variant calling: Aligned reads are then compared to the reference sequence to identify potential variations. Statistical methods differentiate genuine discrepancies (such as structural variations) from sequencing errors, ensuring the reliability of variant calls. This step reveals possible genetic alterations that may be relevant to the phenotype of a disease or a treatment response.
  • Annotation: Enriches identified variants with additional information beyond their mere presence. This includes pinpointing the location within a gene, predicting potential functional consequences (e.g., missense, nonsense mutation), and identifying any known associations with specific diseases or phenotypes. This annotation process transforms raw variants into interpretable findings, aiding physicians in understanding their potential clinical significance and guiding patient management.

Revolutionizing Diagnosis

The impact of NGS extends far beyond tumor biology. It is improving cancer diagnosis by:

  • Offering more accurate and earlier detection: NGS can detect even low-level mutations that can be missed by traditional methods.
  • Identifying specific tumor mutations: NGS paves the way for targeted therapy selection by locating the exact mutations driving tumor growth, offering a more precise and effective approach than broad-spectrum treatments.
  • Classifying and subtyping cancers: NGS helps classify and subclassify cancers based on their unique genetic profiles.

Guiding Treatment Decisions

NGS goes beyond diagnosis by making personalized treatment possible. By profiling a tumor's genetic makeup, NGS allows us to:

  • Match patients to the right therapy: Identify patients most likely to benefit from a specific targeted therapy based on their tumor.
  • Develop personalized treatment plans: Tailor treatment plans to each patient's unique cancer, potentially leading to better outcomes with fewer side effects.
  • Predict and manage drug resistance: Identify mutations associated with resistance to specific therapies.

Beyond Diagnosis and Treatment

The potential of NGS in pathology extends far beyond diagnosis and treatment:

Monitoring Treatment Efficacy

NGS can be used to track tumor response to treatment and detect early signs of recurrence, allowing for adjustments to treatment plans.

Identifying Individuals at Risk

Germline testing through NGS can identify individuals harboring hereditary cancer predisposition mutations, allowing for targeted surveillance programs and early intervention strategies.

Developing New Therapies

NGS accelerates the discovery and development of novel therapeutics by unraveling the genetic underpinnings of tumors. It plays a key role in developing targeted and immune checkpoint therapies, optimizing drug design, and patient selection for clinical trials.

Liquid Biopsies

NGS analysis of circulating tumor DNA (ctDNA) in blood offers a non-invasive way to monitor tumor progression and response to treatment, especially in sensitive or difficult-to-access tumors. This technique holds significant promise for:

  • Monitoring tumor progression and treatment response: This is valuable for sensitive or difficult-to-access tumors. Liquid biopsies enable serial monitoring without the need for repeated tissue sampling.
  • Detecting minimal residual disease (MRD) and early relapse: By identifying ctDNA shed by residual tumor cells, liquid biopsies can potentially lead to earlier intervention and improve clinical outcomes.

NGS analysis of ctDNA provides valuable insights into tumor biology, paving the way for personalized medicine. By identifying relevant mutations, ctDNA analysis can guide targeted therapy selection and monitor mutation dynamics over time, potentially leading to more precise and individualized treatment strategies.

The Future of NGS in Pathology

The future of NGS in pathology is extremely exciting. Advancements in technology are continually increasing the speed, accuracy, and affordability of NGS, making it even more accessible for routine clinical use. Additionally, research is ongoing to:

  • Develop new NGS panels: Target specific cancer types and subtypes for even more precise diagnoses and treatment recommendations.
  • Integrate NGS with other technologies: Combine NGS with other data sources, such as artificial intelligence, to improve diagnostic accuracy and treatment decision-making.
  • Address ethical concerns: The goal is responsible and fair access to NGS testing and to address any potential privacy concerns surrounding genetic data.


NGS offers insights into cancer biology and paves the way for personalized medicine. As NGS technology continues to evolve, its impact on cancer diagnosis, treatment, and ultimately, patient outcomes will be even greater.