There comes a point during the development of every NGS assay at which you want to make sure it will reliably detect everything you say it can. You want its real-world performance to match your claims.
That means fine-tuning your development protocol — from specimen handling, to nucleic acid extraction, to library prep — against a range of variants and allele frequencies to make sure your early-stage assay picks them all up.
Only after doing so can you move the assay on to the costly validation phase.
Naturally, to replicate real-world scenarios, many developers instinctually turn to real patient samples.
But the truth is, if you’re only subjecting your assay to the patient samples your lab has on-hand — or even ones you procure from colleagues or biobanks — you’re not exposing your assay to a wide enough range of variants and conditions to ensure its performance. Plus, you may be costing your lab money and time:
- Development delays can allow another lab to launch their assay before yours.
- Not using robust enough reference materials can lead to your assay not meeting performance claims in production.
While there is clearly a need to evaluate assays with real specimens tested on orthogonal methods, in the development phase multiplexed truth sets such as biosynthetic NGS reference materials are superior to patient specimens.
Here are three reasons not to trust remnant patient samples alone for assessing your clinical NGS assay’s workflow.
1. Patient Samples Are Hard to Come By
Unless your institution is fortunate enough to have its own large-scale biobank, you’ve likely experienced the pain of searching for suitable remnant specimens. And regardless of the size of your lab, everyone has trouble tracking down rare variants.
All this waiting and effort spent searching for variants eats up time and money. Although you can’t validate everything, for the highest confidence before your assay goes into production, you still need to look at as many variant types and technically challenging variants as possible. Too many false negatives could render your assay useless.
2. Patient Samples Are Limited in Quantity
If you want to truly assess the performance of your assay under as many conditions as possible, you’re going to need enough quality reference material to both design your assay and perform a comprehensive validation.
Unfortunately, there is a finite supply of patient specimens bearing clinically relevant mutations. Sometimes, no matter how hard you look, you can’t find the rare variants you need. They are, after all, rare for a reason.
If you do get your hands on some rare material, it’s worth its weight in gold. You may want to save it for the validation phase.
3. Patient Samples Are Unreliable and Noisy
You want your assay to perform the same way, time and time again. In other words, you want your results to be reproducible. Otherwise, how can you trust them?
Before you comprehensively validate your assay with a variety of reference material types, it’s critical to establish its performance with highly consistent, truth-set material. With real patient material, variation inevitably creeps in. Sandi Deans, Consultant Clinical Scientist and Director of UK National External Quality Assessment Service (UK NEQAS) for Molecular Genetics, discussed the inherent variability of patient reference material in a recent webinar.
Dr. Deans shared DNA extraction and quality data that revealed the wide range of results the same sample can generate. She went on to say that, despite the inherent variability of clinical genomics assays, the best way to ensure confidence in the diagnostic result is to use a robust reference material truth set.
A More Reliable, More Abundant Alternative to Patient Samples
So if real patient samples aren’t ideal for testing your assay, what is? Perhaps non-intuitively, biosynthetic NGS reference materials can come closer to mimicking real-world conditions than real patient samples.
The Association for Molecular Pathology and the College of American Pathologists recently published “Guidelines for Validation of Next-Generation Sequencing–Based Oncology Panels.” In it, they wrote:
“[S]ynthetic DNA fragments...have particular advantages because they can be designed to incorporate specific sequence variants at known positions. They, likewise, can be mixed in known allelic ratios, to simultaneously evaluate many aspects of platform performance, library preparation, and bioinformatics analysis.”
In other words, biosynthetic material allows you to test your assay in as many different conditions, for as many different variants as you need. No need to waste time and money hunting down rare variants, or worse, having to avoid validating rare variants altogether.
And If you run out, you can just purchase more, confident that it remains the same from lot to lot. In fact, because biosynthetic NGS reference materials have known variants at precisely quantitated allele frequencies, they can reveal subtle performance issues that could be masked by the innate variability of natural specimens. Even better, these materials can be custom engineered to include the exact quantities and variants needed for a given project.
Why Patient-Like Contrived Samples Improve Your Assay
Biosynthetic NGS reference materials are available in a variety of formats, such as purified DNA mixes to speed development, and full-process reference materials (in plasma and FFPE) to challenge your entire workflow. Remnant patient specimens don’t give you that flexibility.
At SeraCare, we have developed and patented a process to stabilize nucleic acids in a synthetic plasma matrix — built from human-derived plasma components. These reference materials act just like real patient samples throughout the entire workflow, including extraction.
In contrast to real patient samples, they’re highly multiplexed with rare, common, and challenging variants at precise allele frequencies. Because they’re manufactured in a GMP-compliant and ISO-13485-certified facility under design controls, they have exceptional lot-to-lot consistency and long-term stability.
To learn more about how high-performance Seraseq™ biosynthetic NGS reference materials can save you time, boost your confidence, and lower your costs, get your free copy of our new white paper by clicking on the banner below.