Top 10 tips for validating your NGS assay

With new advances in technology, we're now moving more towards large panel next-generation sequencing (NGS) assays and whole exome sequencing (WES). NGS is gaining popularity thanks to high-throughput capability and reduction in cost per sample.

However, validating these complex assays can be tricky, so we've compiled some top tips from industry guidelines to help you.


  1. Using validation samples - also referred to as reference samples, reference material or reference standards - help you understand any variabilities and identify potential errors in your workflow
  2. Validate your assay by using a range of well-characterized validation samples to understand the performance of the test for a given samples type, variant type, genomic region or allele burden and how that can be extrapolated to other sample types, variant types, genomic regions or allele burdens
  3. If the sample type you are using can be problematic (e.g. FFPE), adding validation samples will help you maintain consistent assay performance even if sample quality or quantity is suboptimal
  4. Try to use validation samples with variant types relevant to the tests intended use, such as: SNVs, Indels, CNVs, translocations and fusions
  5. It's important to include at least two well-characterized samples that have known sequences for all regions you're targeting. This will reduce systematic errors that can occur based on regional variations - such as repetitive sequence or pseudogenes
  6. You can use reference samples that are in the same format as your test sample - such as FFPE, or cell-free DNA in synthetic plasma. This means the reference sample will more closely mimic the material you're using in your test. This will help you detect any systematic errors that are likely to occur because of the sample format
  7. Reference standards that are derived from cancer cell lines are an inexhaustible source of material so they are a particularly useful source of reference material for NGS assays
  8. Choosing a reference standard with different variants and different allelic frequencies is an easy way to test sensitivity so that you can ensure the detection of variants at the lowest limit of detection (LLOD)
  9. Reference samples should be used when setting up a new assay, validation, routine monitoring and when a change to the workflow is introduced. For example, introducing a new gene to an existing panel requires revalidation to make sure that any new sequence variations can be detected without compromising assay quality
  10. Horizon has a wide range of reference standards to help you validate your NGS oncology assay, including OncoSpan - the world's largest oncology reference standard, specifically designed for large gene panel NGS and WES, now available in three formats cell-free DNA, FFPE curls, and genomic DNA for use as a whole process control. Simply select your panel and we'll show you which reference standards cover your genes of interest
Check out Horizon's NGS products:
Learn more about Horizon's reference standards
  • The five quality control metrics every NGS user should know – Blog article
  • How Horizon Discovery’s reference standards are made (and how they are right for your assay) – Blog article
  • Using reference standards in the development of new liquid biopsy platforms – Blog article
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  1. Jennings et al: Guidelines for Validation of Next-Generation Sequencing - Based on Oncology Panels. J Mol Diagn 2017, 19: 341-365
  2. Kim et al: Good Laboratory Standards for Clinical Next-Generation Sequencing Cancer Panel Tests. J PatholTM 2017, 51: 191-204