This technical note from Beckman Coulter Life Sciences presents data from a study in which EGFR L858R standards with varying allele frequencies were spiked into plasma and isolated using either Apostle MiniMax High-Efficiency cfDNA Isolation or another extraction product, showing extraction efficiency to affect NGS results.

Due to its small size and low quantity, the main challenge for cell-free DNA analysis is to get enough cfDNA for sequencing. This results in a need to extract from larger volumes of bodily fluid, yet higher input volumes can be more difficult to manage for high-throughput sample processing.

This application note from Beckman Coulter Life Sciences demonstrates the use of the Apostle MiniMax High-Efficiency cfDNA Isolation Kit in conjunction with the KingFisher Duo Prime automated protein purification system to mitigate some of the challenges of processing large-volume samples for cell-free DNA sequencing.

As a non-invasive way to detect disease, cfDNA is extracted from blood; however, there is some concern that cfDNA does not contain the same biomarkers as tumor tissue. Tumor tissue is typically removed and stored as formalin-fixed, paraffin-embedded tissue, a process that preserves the morphological structures well but chemically modifies and degrades the nucleic acids. Despite the difficulties, FFPE tissue is often used to look for cancer-associated mutations; however, it does not always correlate with the mutations seen in cfDNA.

This poster from Beckman Coulter Life Sciences presents a comparison of matched FFPE and plasma samples to determine how many mutations are seen in both tissues and where the mutational mismatches appear in the chromosome, finding that chromosomal regions have different mismatch rates and giving clues about the best chromosomal locations for biomarkers.

This white paper from Beckman Coulter Life Sciences presents data generated by the Apostle MiniMax High-Efficiency cfDNA Isolation Kit, a cell-free DNA (cfDNA) isolation reagent kit built on magnetic bead-based technology demonstrated to purify cfDNA from human plasma in both manual and automated workflows.

With recent advancements in nucleic acid-based molecular technologies, applications for precision medicine in clinical research have grown exponentially. In particular, analysis of cell-free DNA (cfDNA) for cancer detection, prognosis, and monitoring has significantly improved patients’ clinical management and outcomes.

The primary challenge with cfDNA-based liquid biopsy techniques is that cfDNA is typically found at very low concentrations in body fluids, with a range of 1 to 50 ng cfDNA from 1 mL plasma. Of the total cfDNA found in plasma, fragments originating specifically from tumors (ctDNA) can represent as low as 0.01 percent. Thus, efficient techniques for cfDNA extraction are essential for ctDNA detection in precision medicine. Two of the most commonly used cfDNA extraction techniques are spin column-based and magnetic bead-based purification.

This technical note from Beckman Coulter Life Sciences describes a study comparing a spin column-based cfDNA purification kit with a magnetic bead-based purification kit, finding the bead-based kit to significantly outperform the column-based kit and highlighting the importance of selecting an efficient cfDNA extraction method for liquid biopsy applications.

During the bioinformatic and clinical processing of NGS data that will result in the final medical assessment, all detected variants must be checked for if and how they are annotated in genomic databases. However, the existence of significant discrepancies in variant reporting and their classification requires additional scrutiny and caution. Some of the existing discrepancies between databases can only be resolved by manually scanning conflicting journals, assessing, and reviewing supplementary materials in the articles, or directly contacting the authors.

This application note from Qiagen describes how to use Human Gene Mutation Database Professional to increase the diagnostic yield in complex neurological disorders including Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis.

Genetic variation in neoplasia can take the form of single or multiple nucleotide variants, gene fusions, amplifications, and/or splice variations. This heterogeneity can pose a challenge in comprehensive profiling to guide therapeutic decision making. The Oncomine Precision Assay on the Genexus Integrated Sequencer is an amplicon-based automated NGS assay that enables the detection of all these genomic variant classes, spanning 50 genes, focused on those with the greatest clinical relevance and targets of many ongoing trials. The complete workflow, from library prep to final report generation, can be accomplished in less than 30 hours for up to 32 formalin-fixed paraffin-embedded samples. OPAGX uses AmpliSeq HD technology, utilizing unique molecular tags for sequencing error correction and increased sensitivity of variant detection.

This poster from Q2 Solutions describes a study in which the Oncomine Precision Assay on the Genexus Integrated Sequencer was characterized at a Durham, NC-based site and then transferred to and validated by a genomics laboratory in Beijing, enabling global harmonization of clinical trial testing.

High-complexity NGS workflows impact the ability to achieve consistent inter-laboratory performance. Often, interlaboratory performance is highly dependent on operator proficiency and institutional systems designed to minimize the variability of testing performance.

This poster from Q2 Solutions describes the analytical performance and reproducibility of an automated next-generation sequencing pan-cancer DNA and RNA assay at two global laboratory sites located in Durham, NC and Beijing, demonstrating interlaboratory accuracy and reproducibility enabling global implementation and harmonization for clinical testing.

Mutations in BRCA1 and BRCA2 tumor-suppressor genes account for a significant portion of hereditary breast and ovarian cancer cases. Among them, large genomic rearrangements (LGRs) involving a loss or gain of partial complete BRCA exon(s) are responsible for up to 27 percent of all BRCA disease-causing mutations identified. However, these LGRs are frequently missed using both PCR-based methods and targeted NGS assays.

Given the difficulty in detecting LGRs, there is a need for a comprehensive BRCA1/2 testing algorithm including reference materials that incorporate pathogenic BRCA1/2 LGRs to support NGS assays that identify these mutations at both germline and somatic levels. Clinical studies have demonstrated that pathogenic BRCA1/2 mutations sensitized patients to platinum-based chemotherapy and PARP inhibitors. Current guidelines increasingly recommend BRCA testing in management and therapy selection. Accurate detection of BRCA1 or BRCA2 pathogenic variants is therefore critical in breast and ovarian cancer therapy as well as clinical management of disease including patients who are eligible for new PARP inhibitors.

This poster from LGC SeraCare describes the development and validation of reference material to support BRCA1 and BRCA2 gene testing, the first to include 10 LGRs, which will be useful in evaluating the ability to detect challenging LGRs and support PARP inhibitor treatment of patients with breast and ovarian cancers.

Epigenetic modifications such as methylation influence cellular differentiation and gene expression. Liquid biopsies are starting to screen for cancer-derived DNA by looking for unexpected epigenetic modifications in circulating cell-free DNA (ccfDNA) that could also be used to assign a tissue of origin to the cancer to direct confirmatory diagnostic procedures. However, because of the low abundance of ccfDNA, obtaining sufficient amounts for assay development, validation, and proficiency testing are difficult. Furthermore, methods of analyzing the epigenetic modifications, such as bisulfite conversion, can damage a significant fraction of the input material; but, failing to carry them out to completion can result in an overestimation of methylation. This makes method optimization very important.

This poster from LGC SeraCare describes the creation and validation of circulating cell-free DNA reference materials with defined amounts of methylation to address the challenges associated with assessing the methylation of circulating cell-free DNA.

Homologous recombination deficiency (HRD) arises due to a defect in DNA repair and serves as an important therapeutic biomarker. NGS assays that measure HRD status via genomic instability are used to stratify ovarian and breast cancer patients and determine eligibility for clinical trials as well as PARP inhibitor and platinum-based therapies. Testing is performed on FFPE biopsied tissue, and, despite the challenges of this sample type and the complexity of analysis, there are currently no commercially available HRD reference materials that enable standardization between assays.

This poster from LGC SeraCare describes the development and validation of reference materials to aid in developing and validating NGS-based HRD assays and for quality control monitoring of genomic instability scores.

Advances in next-generation sequencing are enabling larger panel sizes and increased sensitivities for liquid biopsies. These advances lead to a need for patient-like reference materials with more clinically relevant alterations at lower levels than before. To address this need, LGC SeraCare generated novel circulating tumor DNA (ctDNA) reference materials that combine much of the content of Seraseq ctDNA reference materials and add additional variants and copy number alterations. Because limits of detection of individual mutations are now at or below 0.2 percent variant allele frequency in some ctDNA assays, SeraCare also reduced the lowest VAF from 0.1 percent to 0.01 percent.

This poster from LGC SeraCare provides data on the initial testing of new circulating tumor DNA reference materials that incorporate updated clinically relevant alterations and copy number variants with the lowest variant allele frequency reduced from 0.1 percent to 0.01 percent to accommodate larger NGS panels with increased sensitivities.

Myeloid malignancies are characterized by excessive proliferation, abnormal self-renewal, and differentiation defects of hematopoietic stem cells and myeloid progenitor cells. The detection of these types of malignancies using traditional procedures by obtaining bone marrow samples is invasive and risky. Hence, liquid biopsy-based methods are in high demand to monitor and detect new cases of blood cancer.

This poster from LGC SeraCare summarizes the development of ctDNA myeloid reference materials — a purified mix of 25 myeloid DNA variants that were blended against a wild-type background — for the development, validation, and clinical application of targeted NGS assays as well as digital PCR-based assays that analyze for myeloid diseases in cancer patients.

This white paper from LGC Biosearch Technologies includes guidance for bringing a molecular diagnostic assay to market, including how to find a partner, ensure component quality, and budget for long-term success.

An important aspect of minimizing the time it takes to get a molecular diagnostics product to market is how product design and the critical components that make up the assay will scale for manufacturing.

This white paper from LGC Biosearch Technologies provides guidance for choosing an experienced supplier for molecular diagnostics components to avoid needing to trial new components in the late stages of product design, redesign an assay, or identify and onboard a new supplier during manufacturing.