Participating in NGS proficiency testing is a requirement for clinically accredited sequencing laboratories and a valuable quality check for any laboratory that wants external confirmation its results are correct. Proficiency testing, also called external quality assessment, works by sending a laboratory samples with answers it does not know, having the laboratory run them as it would patient samples, and comparing the reported results against the expected answers and against peer laboratories. It is the one quality measure that is genuinely external, and that is what gives it force: a proficiency-testing result is visible outside the laboratory, to the scheme provider and the accreditor, so both a good result and a failure carry weight the lab’s internal quality controls do not.
This guide covers proficiency testing for sequencing end to end: the schemes available for NGS assays, how to choose a program by assay type, what to do when no formal scheme exists for your assay, and, most importantly, how to investigate and respond to a failure, because the response is what accreditation actually turns on. The analytical validation that proficiency testing complements is covered in Validating an NGS Assay for Clinical Use.
Key Takeaways
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Available Schemes for NGS
Formal proficiency-testing schemes for sequencing have grown substantially as clinical NGS has matured, and the coverage is now good for common applications even if it remains incomplete for the full breadth of what sequencing can do. In the United States, the College of American Pathologists operates the most extensive set of molecular and NGS proficiency programs, including surveys for next-generation sequencing in solid tumor oncology, multigene panels, and specific clinically important genes, alongside programs for germline testing. Internationally, established providers such as the quality networks that grew out of European molecular genetics quality assessment offer NGS external quality assessment schemes for both germline and somatic testing, and other molecular quality-control organizations run relevant programs, so a laboratory in most regions has access to at least some formal scheme for common sequencing applications.
The trajectory matters as much as the current state: proficiency testing for sequencing is actively expanding to cover more applications, more gene targets, and increasingly the analytical breadth of exome and genome-scale testing rather than only targeted panels. A laboratory setting up a clinical NGS assay should check the current offerings of the major providers for a scheme matching its specific application, because what was unavailable a year or two ago may now exist.
Choosing a Program by Assay Type
The right proficiency-testing program is the one that matches what your assay actually does, because proficiency testing only tests quality where its samples and expected answers align with your assay’s intended use. A laboratory running a solid-tumor somatic panel needs a scheme built around somatic variant detection in tumor material; a germline hereditary-disease laboratory needs a germline scheme; the variant types, the sample matrix, and the reporting all have to correspond to the clinical assay for the proficiency test to be meaningful. Enrolling in a convenient scheme that does not match the assay produces a result that does not actually assess what the laboratory reports clinically.
Assay Type | Match a Scheme That Tests | Where None Fits Exactly |
Somatic / solid tumor panel | Somatic variant detection in tumor material | Alternative assessment, or the closest partial scheme |
Germline / hereditary panel | Germline variant detection in the relevant genes | Alternative assessment for uncovered genes |
Exome or genome | Broad variant detection across the target | Emerging broad schemes, or alternative assessment |
Specialized or novel assay | The specific variants and matrix used | Alternative assessment, usually required |
Table 1. Match the proficiency-testing scheme to what the assay actually detects, in the sample matrix it uses. Where no formal scheme fits, an alternative performance assessment is required rather than optional.
Alternative Assessment Where No Scheme Exists
Sequencing can do more than the available proficiency schemes cover, and when a laboratory runs an assay for which no formal scheme exists, the obligation to assess performance externally does not disappear, it changes form. Accreditation requires that a laboratory assess the performance of every test it reports, so for an assay with no formal proficiency program the laboratory must perform an alternative assessment, typically at least twice a year, to demonstrate the assay continues to perform correctly. This is not a lesser substitute grudgingly accepted; it is a recognized and required part of quality assurance for the many valid assays that outpace the formal schemes.
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Alternative assessment takes several accepted forms: exchanging samples with another laboratory running a comparable assay and comparing results, splitting samples and testing them by an independent reference method, or blind-testing well-characterized reference materials with known answers as if they were unknowns. Each achieves the essential purpose of proficiency testing, an objective external check that the assay produces correct results, without a formal scheme. The laboratory should define its approach in advance, document it as rigorously as it would a formal proficiency test, and treat the result with the same seriousness, because to an accreditor an alternative assessment carries the same weight, and the same consequences if it reveals a problem.
Investigating a Failure
A proficiency-testing failure is where the real quality work happens, and the laboratory’s response, not the failure itself, is what determines the outcome with an accreditor, because a failure handled well demonstrates a functioning quality system while a failure handled poorly or ignored demonstrates the opposite. When a laboratory receives a failing or discordant proficiency result, the first obligation is to investigate promptly and systematically to find the cause: reviewing exactly what was reported against the expected answer, examining every step of how the proficiency samples were handled and analyzed, and determining whether the failure reflects a genuine problem with the assay, a clerical or reporting error, a problem specific to the proficiency samples, or a limitation in the scheme itself.
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The investigation has to distinguish, in particular, between a true analytical failure, where the assay actually would have produced a wrong answer on a patient sample, and other causes such as a transcription error in reporting or an artifact of the proficiency material, because the significance and the required response differ sharply. A true analytical failure raises the question of whether patient results may have been affected and demands the most serious response; a reporting error is still a real finding requiring correction but points to a different fix. Either way, the investigation must be thorough, documented as it proceeds, and aimed at the actual root cause, because the corrective action that follows can only be as good as the investigation it rests on.
The Response Is What Is Assessed, Not the Failure Every laboratory will eventually have a proficiency-testing failure or a discordant result; the schemes are designed to be challenging, and a perfect record over time is less common than outsiders assume. What separates a laboratory that handles this well from one that does not is entirely the response. An accreditor examining a past failure is not asking merely whether the laboratory failed but whether it detected the failure, investigated it competently, found and fixed the cause, and documented the whole process, promptly and within the window the accreditor sets. A failure met with a prompt, thorough, documented corrective action is evidence of a working quality system. A failure ignored, minimized, or poorly investigated is the actual problem. Treat every failure as the start of a corrective-action process, not as an embarrassment to be downplayed. |
Corrective Action and Documentation
Corrective action is the structured response that turns an investigated failure into a resolved one, and it follows the same logic as any corrective-and-preventive-action process: identify the root cause, take the action that fixes it, verify the fix worked, and, where the cause could recur, take preventive action to stop it happening again. For a proficiency-testing failure, that means not only correcting whatever produced the wrong result but confirming, before resuming or continuing to report affected testing, that the assay now produces the correct result, often by re-testing the proficiency material or characterized samples. Where a true analytical failure may have affected patient results already reported, the corrective action extends to assessing and, if necessary, addressing that impact, which is among the most serious obligations a failure can trigger.
Documentation is what makes the entire response real to an accreditor, and a corrective action that was performed but not documented is, for accreditation purposes, indistinguishable from one that never happened. The record should capture the failure, the investigation and its findings, the root cause, the corrective and any preventive action taken, the verification that it worked, and the dates throughout, assembled as the evidence that the laboratory identified and resolved the problem within the required timeframe. This is one instance of the broader corrective-action discipline that quality systems require, developed as part of the accreditation framework inCLIA, CAP, and ISO 15189: Accreditation Requirements for Sequencing Labs, and within the wider quality picture in Quality and Compliance in NGS Labs: From Research Use to Regulated Testing. Handled this way, a proficiency-testing failure becomes what the quality system is designed to produce: a problem found, fixed, and documented. How this fits the whole operational picture is in Next-Generation Sequencing in the Lab: A Manager’s Guide to Building, Budgeting, and Scaling NGS Capacity.
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