How to Complete the CAP NGS Worksheets: NGS Validation Step by Step
The College of American Pathologists publishes a set of free NGS worksheets, developed with AMP representation and later incorporated into CLSI guideline MM09, that walk a lab through the life cycle of a clinical NGS test with a focus on germline applications. Source: CAP NGS Worksheets. This page is an independent companion written by SLC. It does not reproduce the worksheets; download them from CAP. Here we explain each stage in practical terms, the gaps we see, and what evidence to keep.
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.
1. Test familiarization
Before writing a single protocol, decide whether NGS is the right method, what clinical question the test answers, which specimen types and turnaround you need, and whether you have the space, staff and budget. Evidence to keep: a signed test proposal with intended use, a feasibility and cost assessment, and a decision on platform.
2. Test content design
List genes, disorders and key variants that make the test clinically valid. Identify difficult regions (high GC, homologous genes, pseudogenes, repeats) and decide how each critical region will be covered, by NGS or by a supplementary method. Choose reference materials for validation. Evidence: target region list with rationale, problem-region map, reference material plan.
3. Assay design and optimization
Translate content into a method: hybrid capture or amplicon enrichment, coverage targets, sequencing configuration, and any supplementary assays such as Sanger or MLPA. Run optimization experiments and record what changed and why. Evidence: optimization data, locked protocol, rationale for coverage thresholds. See our library preparation guide.
4. Test validation
Establish accuracy, precision, analytical sensitivity and specificity, and reportable range by variant type, as required by 42 CFR 493.1253(b)(2) for lab-developed tests. Use the worksheet calculators for confidence intervals. Evidence: validation plan approved before testing, raw data, summary report with acceptance criteria, signed by the director. See NGS assay validation.
5. Quality management
Define monitors across pre-analytic (specimen acceptability, DNA quantity and quality), analytic (library yield and size, run metrics, coverage, controls) and post-analytic phases (report review, turnaround, amended reports). Evidence: QC SOP with lab-defined acceptance criteria, trend logs, corrective actions. See run QC metrics.
6. Bioinformatics and IT
Document compute infrastructure, each pipeline component and version, parameters, reference genome build, annotation sources, security and storage. Validate the pipeline and revalidate on change. Evidence: pipeline description, version log, validation data, change control records. See pipeline validation.
7. Interpretation and reporting
Set variant filtration, classification approach (for germline, ACMG/AMP criteria are widely used), handling of incidental and secondary findings, reclassification and reanalysis policies, and report content including limitations and regions not covered. Evidence: interpretation SOP, report template, reclassification log.
Common gaps inspectors find
Validation plan written after data were collected
Acceptance criteria not defined for each variant type
Pipeline version used clinically differs from validated version
No documented revalidation after a software or kit update
Coverage gaps not disclosed on reports
Missing competency records for bioinformatics steps
Alternative PT not performed twice yearly
How SLC helps
SLC helps labs work through each stage: drafting test proposals, designing validation studies, writing QC and bioinformatics SOPs, building report templates, and preparing evidence binders for CAP inspection. SLC is not affiliated with CAP, AMP or CLSI.
Before you start: assemble the team and the binder
The worksheets assume input from several roles: the laboratory director or designee who owns the test, a molecular technologist who will run the bench work, a bioinformatician, and the person who will interpret and sign reports. Assign an owner for each stage and set up a validation binder or electronic folder with one section per stage. Every decision you make should land in that folder with a date, the decision maker and the rationale. Inspectors look for that trail.
Turning worksheet answers into SOPs
The worksheets are planning and reasoning tools, not procedures. Once a stage is complete, translate the outcome into controlled documents: a test procedure, a QC procedure, a bioinformatics procedure and a reporting procedure, each approved by the director before patient testing. Keep the completed worksheets as supporting records that explain why the SOP says what it says.
Validation statistics in plain terms
Positive percent agreement tells you what fraction of true variants your test found. Positive predictive value tells you what fraction of the variants your test reported were real. Both need confidence intervals, because a perfect score on a small sample set does not prove high performance. Report results separately for single nucleotide variants, small insertions and deletions, larger indels, copy number changes and any other class you claim, and for difficult regions separately from easy ones.
Reference materials that hold up
Use a mix of publicly characterized reference genomes, commercially available engineered reference materials with known variants, and previously tested clinical samples with results from an orthogonal method or another accredited lab. Document the source, lot, and truth set version for each. Where truth data are incomplete, state that limitation in the validation report.
After go-live: the worksheets keep working
The quality management and bioinformatics stages describe ongoing work. Trend QC metrics, review failed runs, track turnaround, and revisit validation whenever you change a kit, enrichment design, sequencer model, pipeline component or reference database. Treat the worksheet structure as the outline for your change control assessments, so each change is evaluated against the same questions you answered during development.
Evidence checklist for inspection day
Signed test proposal and intended use
Target region list with problem-region analysis
Optimization records and locked protocol
Approved validation plan dated before data collection
Validation data, summary and director signature
QC SOP with acceptance criteria and trend logs
Pipeline documentation, version log and change control
Interpretation SOP, report template, reclassification log
PT results or alternative assessment records
Training and competency records for every role
Applying the worksheet approach to somatic and other tests
The CAP worksheets focus on germline testing and CAP lists somatic content as in progress. Labs building tumor panels can still follow the same life-cycle logic, adding somatic-specific steps: tumor content review by a pathologist, limit of detection by variant allele fraction, FFPE artifact handling, matched or unmatched normal strategy, and therapy-oriented reporting tiers. Document where your approach departs from germline-oriented guidance and why.
A practical timeline of stages
Stages overlap in practice. Familiarization and content design usually happen together; assay optimization feeds back into content when coverage of a key region turns out to be poor; and bioinformatics design should start during optimization, not after validation. Plan the validation study only after the assay and pipeline are locked, because any later change to either means repeating work. Quality management and reporting procedures should be drafted before validation ends so they can be tested on validation samples.
Mistakes that cost the most time
The most expensive errors are locking a sequencer or enrichment design before defining clinical content, starting validation before the pipeline is frozen, collecting too few samples for the variant types claimed, and writing the report template last. Each forces repeat work. A short design review at the end of each stage, signed by the director, catches most of them.
Who signs what
The laboratory director is responsible for approving the test, the validation plan, the validation report, procedures and changes. Delegation is possible for some tasks under CLIA and CAP rules, but the director must document delegation and stays accountable. Keep signatures and dates on every key document so the sequence of decisions is clear.
Linking worksheets to the CAP checklist
During inspection, the CAP checklist, not the worksheets, is the measuring stick. Build a crosswalk that maps each relevant checklist requirement to the document or record that satisfies it, many of which will come from your worksheet work. Update the crosswalk whenever CAP releases a new checklist edition.
Using the worksheets for an existing test
Labs that launched NGS before the worksheets existed can use them as a gap assessment. Walk through each stage, compare what the worksheet asks with your existing records, and list missing items. Many labs find gaps in problem-region analysis, documented rationale for coverage thresholds, and bioinformatics change control. Close gaps with addenda to the validation report or new procedures rather than rewriting history, and date everything.
Keeping the work current
Review the worksheet-based documentation at least when CAP publishes updates to the worksheets or checklist, when CLSI revises related guidance, and when you change the test. Assign an owner for each section and record the review date so the file stays inspection-ready.
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
Are the CAP NGS worksheets free?
CAP publishes them on its NGS worksheets page; check CAP's site for current access terms.
Are the worksheets mandatory?
They are educational guidance, not checklist requirements; CAP describes them as concepts and teaching examples rather than prescriptive standards.
Do they cover somatic testing?
They focus on germline; CAP lists somatic content as in progress.
Can SLC complete them for us?
SLC helps your team complete the work and assemble evidence; your director remains responsible.
Sound Lab Consultants (SLC), PO Box 2135, Chandler, AZ 85244. A nationwide clinical laboratory consulting network. Plans from $500.