Optimizing NGS Sample Quality for Cancer Research

Next-generation sequencing (NGS) has played a significant role in advancing cancer research by identifying oncogenic mutations, uncovering transcriptome variations, and providing insights into the tumor microenvironment. However, cancer research often relies on difficult sample types, and obtaining valuable sequencing data from these samples presents numerous obstacles. Despite these challenges, a range of techniques and technologies, supported by expert advice, can optimize NGS sample quality and contribute to the advancement of cancer research.

Sample types and challenges

“Each sample type has its challenges, starting with tissue samples where collection requires an invasive procedure, which can be both painful and expensive,” explained Bill Hunt, Director of Product Management (Genomics) at Standard BioTools™. “Additionally, multiple samples from the affected tissue may be needed to generate a representative picture of the condition.” Hunt then highlighted several particularly challenging sample types. For instance, blood samples, which are rich in proteins, can complicate biomarker measurements and often require specific protocols for collection, storage, and handling. Other samples like circulating tumor cells (CTCs) present a unique challenge due to their low abundance in the bloodstream.

Hunt also mentioned that peripheral blood is a valuable source for biomedical and clinical research, but certain challenges remain in using whole blood for biomarker discovery. While it was typically believed that the most important biomarkers were found in the fraction of whole blood that contains peripheral blood mononuclear cells (PBMCs), isolating PBMCs for this purpose is difficult and expensive, and can alter their transcriptional profile. New devices can stabilize this profile but lysed cells prevent separating PBMC nucleic acids from unwanted material, thereby complicating analysis. Furthermore, Hunt pointed out that globin transcripts can dominate red blood cell fraction and may constitute up to 70% of RNA in whole blood. This prevalence of globin transcripts can significantly overshadow other reads from RNA sequencing libraries and adds another layer of complexity to the analysis.

Michael Ruvolo, R&D Director of NGS Assay Development at Agilent, highlighted that tumor biopsies are crucial for cancer research, but remain challenging due to the nature of the collection process and the limited availability of samples. He pointed out the issues with FFPE tissues and liquid biopsy/plasma samples, noting “the difficulty with FFPE tissues lies in the preservation method, which can damage DNA and RNA, significantly lowering the quality and recovery amount.” Moreover, the high susceptibility of RNA to degradation complicates the process even further. For liquid biopsy samples, Ruvolo shared that they pose a challenge due to the minimal presence of cell-free DNA (cfDNA) and the degraded state of this DNA. For both types of samples, there is often only one opportunity for nucleic acid extraction, making these samples highly valuable.

Jonathan Bibliowicz, Cancer Genomics Associate Director at PacBio, underscored the obstacles faced in cancer research, particularly due to traditional sample preservation and extraction methods. He emphasized that with the shift toward long-read sequencing in cancer genomics, obtaining high-quality high molecular weight (HMW) DNA is necessary to recognize the full potential of discovery from a sample. However, legacy techniques such as FFPE, often result in degraded samples leading to suboptimal results. This challenge has created the demand for dedicated sample extraction protocols that optimize DNA quality, reduce fragmentation, and produce successful sequencing results.

Overcoming sample challenges

Innovative solutions now enable researchers to overcome many of these challenges and improve the quality of NGS samples in cancer research. For whole blood total RNA samples, Hunt recommended using commercially available globin-reduction protocols in combination with tools like the Advanta™ RNA-Seq NGS Library Prep Kit, due to its high genome mapping rates and low reads for unwanted RNA. When working with frozen whole blood samples, employing quick thaw procedures can reduce RNA degradation as well as increase the yield and quality of extracted nucleic acids. To further protect the DNA, Hunt advised gentle handling of the samples to avoid hemolysis, along with minimizing exposure to light or warm temperatures, which can also introduce damage.

Due to the challenging nature of FFPE samples from the formalin fixation process, Hunt instructed researchers to carefully choose their DNA extraction protocols and emphasized the importance of including purification steps and extended Proteinase K digestion for better RNA yield. Furthermore, Hunt explained that CTCs are indicative of cancer prognosis and tumor diversity, and require various methods for their enrichment and analysis. Their presence in blood samples varies with the tumor’s shedding rate and inherent heterogeneity, affecting the detectable CTC markers. However, emerging sampling methods, including malignant pleural and peritoneal effusions, improve sourcing for CTCs, despite often yielding low DNA amounts. Considering the scarcity of DNA, researchers can utilize microfluidics-based protocols for library preparation to preserve their valuable specimens for additional testing.

Another key strategy involves selecting HMW extraction methods that ensure the successful recovery of nucleic acids for a variety of sample types, including human tissue and frozen blood. Bibliowicz explained that a notable innovation in this area is the Nanobind extraction method, which employs magnetic discs coated with a dense layer of micro- and nano-structured silica. This technology preserves fragment lengths and produces high-purity DNA ranging from 50 to over 300 kb through a rapid magnetic purification process. More recently, a Nanobind PanDNA kit was introduced and enables cancer researchers to investigate multiple sample types.

Ruvolo also stressed the need to choose the right extraction method or kit to ensure high yield and quality, especially for DNA or RNA from FFPE samples. Along with these recommendations, Ruvolo highlighted the importance of using a trusted targeted library preparation method that can handle a low input of starting DNA or RNA, including kits based on the latest Agilent SureSelect chemistries, to generate high-quality libraries. For challenging sample types such as cell-free DNA samples, Agilent Avida technology can capture both DNA variants and methylation alterations from a single sample (no splitting of samples is needed). These methods can be further improved through automation that ensures consistency between samples.

Finally, Ruvolo shared that tracking the quality/integrity of DNA or RNA by performing quality control (QC) analysis steps during the NGS workflow is another important step. QC metrics can provide valuable insights to determine if the sample and library quality and quantity are adequate to continue to the next steps. For example, the %cfDNA score, can help to distinguish the amount of sample processable for downstream experiments from high-molecular weight contaminations. Using automated electrophoresis systems like the Agilent Fragment Analyzer, TapeStation, and Femto Pulse, researchers can effectively monitor the integrity of DNA and RNA with minimal sample volumes. The Femto Pulse system, Bibliowicz noted, also comes highly recommended by PacBio as the top method for accurately sizing long DNA fragments and quality-checking samples from extracted gDNA to HiFi libraries.

While numerous sample types and preservation methods make cancer research more complex, these innovative solutions and advanced technologies can improve this process and help researchers optimize the quality of their samples and perform successful NGS experiments.