NGS For Precision Oncology:
Cancer Next Generation Sequencing

Enabling Cancer Precision Medicine and Research via Rapid and High-Quality Amplicon Sequencing

According to WHO, 9.6 million people worldwide are estimated to have died from cancer in 2018. On the molecular level, cancer is a disease originating from malignant DNA mutations and subsequent changes of gene expression patterns that trigger uncontrolled cell division. In fact, two cancers with the same histologic origin may be different in terms of disease progression and treatment response. These differences result from the unique genetic content of each cancer. Therefore, understanding the genetic makeup of cancer specimens is critical for elucidating the underlying biology.

Next-Generation Sequencing (NGS), especially targeted sequencing, has allowed for the genetic profiling of individual patient tumors, enabling doctors to tailor therapies based on their patient’s specific cancer mutational profile. Hybrid capture and multiplex PCR are the two main targeted sequencing methods for precision oncology. Due to its sensitivity and workflow issue, hybrid capture-based sequencing can be less effective in dealing with samples containing either a low amount of DNA or low-frequency cancer mutations among normal DNA backgrounds. Compared to hybrid capture, PCR amplicon sequencing could be a better choice mainly because it is highly sensitive, cost-effective, and easy to implement in routine laboratory use.

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CleanPlex DNA Technology is a patented, scalable, and ultra-sensitive multiplex PCR-based targeted DNA sequencing technology for next-generation sequencing (NGS). CleanPlex DNA workflow (Figure 1) starts with extracted DNA from fresh frozen tissue samples, FFPE tissues, cancer liquid biopsy samples, etc. The streamlined protocol can be completed in just 3 hours. Scientists or clinicians have the option to add molecular barcodes (unique molecular identifiers – UMIs) into amplicon libraries for more accurate detection of low-frequency variants via CleanPlex UMI Technology.

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CleanPlex® DNA and RNA Amplicon Sequencing Technologies – Simple, Affordable Solution for Cancer Precision Medicine and Research

CleanPlex DNA Technology is a patented, scalable, and ultra-sensitive multiplex PCR-based targeted DNA sequencing technology for next-generation sequencing (NGS). CleanPlex DNA workflow (Figure 1) starts with extracted DNA from fresh frozen tissue samples, FFPE tissues, cancer liquid biopsy samples, etc. The streamlined protocol can be completed in just 3 hours. Scientists or clinicians have the option to add molecular barcodes (unique molecular identifiers – UMIs) into amplicon libraries for more accurate detection of low-frequency variants via CleanPlex UMI Technology.

    

Ready-To-Use Cancer Panels

To meet the needs of different customers, Paragon Genomics has developed a series of ready-to-use panels including:

Learn More About NGS Read-To-Use Precision Oncology Panels

CleanPlex Applications in Oncology

Genomic Profiling of Hematologic Malignancies:

Prof. Elliot Stieglitz’s Lab at the UCSF Benioff Children’s Hospital developed a 25-gene custom NGS assay powered by Paragon Genomics’ CleanPlex technology to retrospectively measure the molecular burden of juvenile myelomonocytic leukemia (JMML) patients at the time of initial diagnosis and posttherapy before hematopoietic stem cell transplantation.

When we decided to choose a target enrichment platform, we had several choices and I am delighted that we chose Paragon Genomics. Paragon Genomics has enabled us to move our sequencing in-house with an affordable and easy-to-use product. They assisted us every step along the way from choosing the amplicons, to guiding us through library preparation to assisting with analysis. I strongly recommend their CleanPlex® amplicon-based targeted sequencing approach and could not be happier with their product.

Elliot Stieglitz, MD, Assistant Professor, University of California San Francisco.

Genomic Profiling of Single Circulating Tumor Cell (CTC):

There is a wide range of instruments and methods for capturing, enriching, and enumerating circulating tumor cells (CTCs). Once isolated, CTCs can be studied using next-generation sequencing (NGS) to obtain their genomic profiles. Targeted sequencing of CTCs using amplicon-based NGS panels is an ideal approach because the workflow is simple to implement and requires little amount of input DNA.

In collaboration with the expert teams at RareCyte and Mayo Clinic, CTCs were visually identified using RareCyte’s CyteFinder® Instrument and were mechanically retrieved with RareCyte’s CytePicker® Module for genomics analysis. Cells were lysed in a PCR-compatible lysis buffer without whole genome amplification, and the lysate was used to prepare targeted NGS libraries using the CleanPlex OncoZoom Cancer Hotspot Panel. This approach resulted in libraries with excellent and consistent coverage and low error frequencies (Figure 3), enabling efficient and accurate assessment of somatic mutations in CTCs.

Tumor Mutational Burden For Immunotherapy Response:

Tumor mutational burden (TMB) is gaining tremendous interest in recent years as a biomarker particularly for predicting response to immuno-oncology (IO) therapies. TMB is measured as the total number of mutations per megabase within the coding region of a tumor genome. Patients with high TMB have been found to have a more favorable response to checkpoint inhibitors, such as atezolizumab and pembrolizumab, than those with lower TMB.

Paragon Genomics has developed a high-performance CleanPlex TMB Panel that contains over 500 genes covered by approximately 27,000 amplicons. One custom version of the panel was tested by Berry Oncology (a subsidiary of Berry Genomics in China) to measure TMB in solid tumor FFPE samples. For each sample, the TMB was calculated, the types of mutations were documented, and the top mutated genes were identified. They compared the mutations identified using the TMB panel to those identified with WES for the same 103 samples and found a high degree of overlap between the two methods. They also found a high degree of correlation when they compared the TMB calculated using the two methods.

CleanPlex Custom NGS Panels

In addition to ready-to-use panels, Paragon Genomics also offers CleanPlex Custom NGS Panels powered by CleanPlex Technology. Researchers and clinicians pick targets or genes that they would like to interrogate, and our NGS bioinformatics experts design and deliver the custom assays in complete kit format (input to sequencing-ready NGS libraries) in just 3 to 4 weeks. Our bioinformatics design team has experience in designing NGS panels for solid tumor cancer types such as lung cancer, breast cancer, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, melanoma, pancreatic cancer, thyroid cancer, etc. and blood cancer types such as leukemia, lymphoma, and myeloma. Our team has also worked on custom panels related to homologous recombination deficiency (HRD), tumor mutational burden (TMB), MET Exon 14 Skipping, etc.

CleanPlex RNA Technology is an ultra-high multiplex PCR-based target sequencing solution for RNA samples. CleanPlex RNA workflow (Figure 2) adds an additional proprietary reverse transcription step in front of CleanPlex DNA workflow so as to convert RNA to cDNA for downstream multiplex PCR amplification. By doing so, CleanPlex RNA inherits all the advantages and features of CleanPlex DNA technologies.

CleanPlex RNA applications include single-cell RNA analysis, gene expression analysis, microRNA, cancer fusion gene detection, etc. Paragon Genomics has launched several CleanPlex RNA-based ready-to-use cancer fusion panels. We also accept CleanPlex RNA Custom Panel requests from researchers and clinicians.

Why Choose CleanPlex NGS Sequencing Panels for Oncology

CleanPlex NGS Panels for Oncology are powered by Paragon Genomics’ CleanPlex Technology, an ultra-high PCR amplicon-based targeted sequencing technology for NGS. As mentioned, after researchers select their targets, our experts deliver the custom assay in complete kit format for effective confirmation of oncology testing.

With Paragon Genomics CleanPlex NGS Oncology Sequencing Panels, you get:

Learn More About Custom NGS Oncology Panels

Quick Turnaround Time (TAT):

Get your personalized NGS panels in less than 4 weeks (from design acceptance to shipment). Easily build a CleanPlex Custom NGS or Sequencing Panel to accelerate your research and assay development.

Use ParagonDesigner™, our free online multiplex PCR primer design tool, to submit your target regions of interest and instantly receive a design coverage report to review. Our custom amplicon panel design experts will be available through the process to provide you a quote, help you with any questions, and make further optimization to meet your needs.

High Performance:

Jumpstart your work with NGS panels tailored to your needs. Our panels are expertly designed with high first-pass success and a high target design rate due to Paragon’s highly advanced proprietary primer design algorithm and an innovative, patented background cleaning chemistry. Together, they allow CleanPlex Custom NGS Amplicon Panels to break the limits of existing target enrichment technologies.

Cost-Effective Sequencing for Oncology:

Generate data with high on-target performance and coverage uniformity to minimize the cost of sequencing.

Ultra-High Multiplexing:

Multiplex up to 20,000+ amplicons per pool for large NGS panels to help you unlock new applications using a faster and simpler workflow for your oncology testing.

Flexible and Scalable Content:

Update your custom NGS Panels as you gather new oncology testing insights.

High Level of Support for Your Oncology Testing:

Receive speedy and comprehensive support throughout the design and ordering process. Our PhD-level expert scientists are here to help create a custom assay for your oncology testing.

NGS for Precision Oncology: FAQs

What is the future of oncology testing?

Cancer often requires a significant amount of time to develop, making it crucial to create and utilize methods for early detection of the disease. Additionally, it’s important to understand the genetic makeup of cancer specimens when creating efficient screenings of the disease that are more proactive than reactive. This is why the future of oncology testing is in Next-Generation Sequencing (NGS). With NGS cancer testing and precision oncology, researchers can learn about the specific genetic makeup of cancer through genetic profiling of tumors. This can help doctors create therapies based on their patient’s specific cancer mutation profiles. CTC Sequencing and Liquid Biopsy are two specific types of Next-Generation Sequencing models that can help with early cancer detection.

Circulating Tumor Cells (CTC) Sequencing: CTCs are released into the bloodstream as a result of cancer and metastasis. They also have the potential to create new metastatic lesions, which are responsible for many cancer-related deaths. The analysis of CTCs through CTC sequencing enables researchers to investigate cancer cell biomarkers and mutational status in a non-invasive manner.

Liquid Biopsy: With the Liquid Biopsy method, researchers can study and detect specific mutations in DNA that come from cancerous cells in plasma, urine, serum, and cerebrospinal fluid. Because liquid biopsy is considered to be non-invasive, it is easy to collect samples from individuals. Liquid biopsy can help with the early detection of cancer, help to create customized levels of more effective treatments and therapies, and identify potential resistance mechanisms.

What types of custom panels can you design?

Array-based approaches to genotyping have historically been the gold standard to analyze regions of the genome for agriculture. However, with the advent and massive development in next-generation sequencing (NGS), researchers have turned to genotyping by sequencing as a method to screen novel regions of interest for plants and animals. For applications such as genetic mapping and variant discovery, genotyping by NGS offers a lower cost per sample.

Is there a minimum number of targets for a custom NGS oncology panel design?

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