Sequencing Controls Improve Confidence in NGS Patient Results

Close Up of DNA Sequencing on a Computer Monitor From a DNA Analysis Machine
[Source: Monty Rakusen/Getty Images]

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Horizon offers diagnostic reference standards to ensure NGS assays are performing at the highest level. We have found optimization and validation of NGS workflows improve when using cell line-derived reference standards that closely represent the patient sample. Our aim is to provide reliable, reproducible, and affordable cell line-derived reference standards to help improve confidence in NGS patient results.

What challenges do you see facing the adoption of NGS panels in clinical testing?

Accuracy in structural variant calling: Structural variants, such as insertions and deletions (INDELs), fusions, single nucleotide variants (SNVs), and copy number variants (CNVs) are often difficult to detect or call correctly. We are starting to see data to suggest that fusions are under-reported by many NGS assays when compared to digital-droplet PCR (ddPCR).

Validation and verification challenges: Accuracy, precision, and sensitivity of the test must be established; ideally at the testing center itself. Many samples are needed to undertake validation and verification experiments but obtaining enough clinical samples with the correct features to interrogate an NGS panel assay is difficult and often there is not enough material for repeat testing of discrepant results. Manufactured reference standards may help, but these controls are often synthetic and may not sufficiently mimic patient sample material.

Results analysis: Bioinformatic algorithms (often referred to as “variant callers”) analyze sequencing data produced by the panels, but also need to be validated and verified. To do this, samples need to be sequenced with high confidence so that the results produced by the variant callers can be compared against a reference control. Due to the variance seen in clinical samples, and the limited amount of material available from a single clinical sample, reproducible reference standards are required. These are often derived from cultured cell lines that have undergone sequencing characterization, such as the NIST Genome in a Bottle (GIAB) cell lines.

Routine monitoring: Errors can be introduced at any stage of the NGS sequencing workflow: during sample extraction, library preparation, sequencing, data processing, data analysis, and data interpretation. It is important to have a whole process control on every run to make sure that the workflow has performed appropriately. Controls can be introduced at various stages of the workflow, enabling easy identification of failure points. This can aid troubleshooting and allow the clinical testing to proceed with minimal downtime.

Using multiple controls can take up valuable patient sample space, increasing turn-around time to providing patients their testing results. Whereas using highly multiplexed controls takes less space, more patient samples may need to be tested on each run.

How can Horizon’s reference standards help with sequencing challenges in the clinic?

No matter the assay or specific challenge a clinical testing facility faces, there is a need for a commutable control that is a close representation of a real clinical sample. We believe that control should be reliable, consistently available, and affordable.

Horizon offers control families that address these challenges in cell-free DNA (cfDNA), genomic DNA (gDNA), and Formalin-Fixed Paraffin-Embedded (FFPE) formats.

OncoSpan—The complete tool for assay validation, verification, and routine monitoring.

Supplied with batch specific whole exome sequencing (WES) data to test your variant caller, it contains 152 genes and 380+ variants present with allele frequencies ranging between 1% and 100%, with 25 of the variants validated using ddPCR.

Quantitative multiplex—Relevant for routine monitoring.

Contains multiple SNPs and INDELs, and includes 11 ddPCR-validated mutations, at 0.8–24.5% allele frequency.

Structural multiplex—For confidence to determine structural variants.

Designed to challenge your workflow by providing validated CNVs, translocations, and large INDELs. The Structural Multiplex includes 9 ddPCR-validated mutations, with most of them centered at 5% allele frequency.

Multiplex I—Perform more robust Limit of Detection (LOD) testing.

Suitable for input pre- and post-extraction to analyze the impact of the liquid biopsy pre-analytical pipeline, Multiplex I is also provided as a set of allelic frequencies across the same variants. Available as cfDNA and cfDNA in plasma.

Contact us to learn more

Horizon’s Reference Standards are manufactured using cancer cell lines that have been engineered to contain or contain endogenously relevant biomarkers at a range of allele frequency. These relevant biomarkers may be used to confirm LOD in your assay and assess sequencing performance. To mimic clinical samples, those cell lines are either embedded into an FFPE block or used to make cfDNA in a plasma-like material. This creates whole process controls for solid tumor and liquid biopsy testing. We also use the cell lines to create gDNA or formalin-compromised DNA (fcDNA) that can be used as a sequencing control. Customers often find multiple formats useful to quickly troubleshoot conditions and verify assay performance.

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