NGS Proficiency Testing and the CAP Checklist
Proficiency testing proves your NGS test gives the right answer on unknown samples. Here is how PT and alternative assessment work for clinical sequencing.
SLC is an independent, nationwide clinical laboratory consulting network. We are not affiliated with, authorized by or endorsed by any sequencing platform maker, kit supplier or accrediting organization.
CLIA and PT for NGS
CLIA Subpart I lists regulated analytes requiring PT; most genetic tests are not on that list. Labs must still verify accuracy of non-regulated tests at least twice a year (42 CFR 493.1236(c)).
CAP expectations
CAP-accredited labs enroll in CAP-accepted PT where available for the analyte or method, and otherwise perform alternative performance assessment at least semiannually. CAP offers NGS PT surveys, including germline and somatic programs and bioinformatics-focused (in silico) challenges.
Alternative assessment options
Split-sample exchange with another lab
Re-testing de-identified previously characterized samples
Reference materials with known variants
In silico data challenges for the pipeline
Handling PT correctly
Test PT samples with patient workflow, don't refer them, keep attestations, review failures with root-cause analysis and corrective action.
CAP NGS checklist topics
Expect inspectors to review wet-bench process documentation, QC metrics and run acceptance, pipeline validation and version control, data storage, variant interpretation, and reports.
Verified regulatory basis
42 CFR 493.1236(c) requires labs to verify, at least twice annually, the accuracy of any test not included in Subpart I and any listed test for which compatible PT samples are not offered by a CMS-approved program. 493.1236(d) requires all PT evaluation and verification activities to be documented. Because most NGS tests are not Subpart I regulated analytes, this semiannual verification is the CLIA floor.
Designing an alternative assessment program
Write a procedure that names the method for each test (split samples, blinded retesting, reference materials, in silico challenges), the number and type of samples per event, how samples are blinded to testing staff, acceptance criteria, who reviews results, and what happens after an unacceptable result. Rotate samples so different genes and variant types are challenged over time.
Covering wet lab and bioinformatics
A good program challenges both. Physical samples test the whole workflow; in silico data files test the pipeline and interpretation. Use both where available.
Investigating PT failures
Treat any unacceptable result as a signal: confirm the result, look for clerical errors, review QC and run metrics, check the pipeline and interpretation, determine root cause, correct, and assess whether patient results were affected and need amendment. Document every step and the director's review.
Competency connection
PT and alternative assessment samples can also serve as blind samples for competency assessment of testing personnel, one of the six CLIA competency elements, when tested as part of routine work.
How SLC helps
SLC helps labs choose PT programs, design alternative assessment procedures, investigate failures and build documentation for CAP inspection. Plans from $500.
Choosing PT programs
Look for programs that match your test type (germline, somatic, hereditary cancer, pharmacogenomics, infectious disease), specimen type, variant classes and reporting style. Programs may supply extracted DNA, cell lines, contrived tissue or data files. Where a program covers only part of your test, combine it with alternative assessment for the rest.
Split-sample exchange
Exchanging de-identified samples with another laboratory that performs a comparable test is a common alternative. Write an agreement covering sample types, timelines, how results will be compared and what counts as agreement. Choose a lab with an established, validated method.
Sample selection for alternative assessment
Use samples that challenge the test: variants in difficult regions, low allele fractions near your LoD, CNVs and indels, and negative samples. Blind the samples so staff test them like patient specimens.
Records to keep
Program enrollment and event schedule
Attestation statements signed by testing personnel and director
Submitted results and program evaluations
Alternative assessment procedures, results and reviews
Investigations and corrective actions
Retention for at least 2 years under 42 CFR 493.1105
CAP inspection preparation
Gather two years of PT and alternative assessment records, investigation files and corrective actions, and be ready to show how each test on your menu is covered.
Somatic and germline differences
Somatic PT challenges usually focus on detecting variants at specified allele fractions in tumor-like material and on correct reporting of clinically relevant findings. Germline challenges focus on detecting and correctly classifying variants across genes, sometimes including copy number events. Choose challenges that mirror what you report.
Interpretation challenges
Some programs score interpretation and classification as well as detection. Where they do not, consider adding internal case reviews or blinded reclassification exercises to your alternative assessment so the interpretive part of the test is also challenged at least twice a year.
Timing and workload
Map PT and alternative assessment events on a yearly calendar alongside competency assessments and calibration or maintenance events. Spacing events evenly avoids last-minute rushes and makes it easier to show inspectors a complete record.
Rules for handling PT samples
CLIA's PT integrity rules apply to NGS too: test PT samples with your regular patient workload by the personnel who routinely test patient specimens, using your routine methods; do not discuss results with other labs before the program's reporting deadline; and never send PT samples or portions of them to another laboratory for testing, even if you would normally refer that test. Document that these rules were followed in the attestation for each event.
What inspectors ask
Expect questions such as how each test on your menu is covered by PT or alternative assessment, how you chose your samples, how you handled any unacceptable result, and who reviewed the results. Clear, organized records make these questions short.
Software: compliance tracking with myLabCompliance
Paper binders and spreadsheets break down once a lab runs multiple instruments, shifts and testing personnel. We recommend myLabCompliance, a web-based CLIA compliance management platform for technical consultants, lab directors, lab owners and quality teams. Its public site describes tools for personnel management, validation, quality control, equipment, document control, scheduling, analytics and audit-ready workflows.
QC logs and quality control records kept in one place
Personnel records and competency tracking
Validation records for new instruments and methods
Equipment records for maintenance and calibration
Document control for SOPs and policies
Scheduling for recurring tasks such as PT events and competency
More NGS resources from SLC
Frequently asked questions
Is NGS PT required?
CAP requires available PT or alternative assessment twice yearly; CLIA requires semiannual accuracy verification.
Can I use in silico PT?
CAP offers in silico challenges for bioinformatics; confirm they fit your test.
Does SLC run PT?
No. SLC helps labs choose programs and document results.
Sound Lab Consultants (SLC), PO Box 2135, Chandler, AZ 85244. A nationwide clinical laboratory consulting network. Plans from $500.