According to the Centers for Medicare and Medicaid Services (CMS), Clinical Laboratory Improvement Amendment (CLIA) registration is required for entities that perform a single test on, “materials derived from the human body for the purpose of providing information for the diagnosis, prevention or treatment of any disease or impairment of, or the assessment of the health of, human beings”.
To date, only two next generation sequencing (NGS) instruments/tests have been approved or cleared by the FDA. All other NGS based tests are developed in house as laboratory developed tests (LDTs), and are regulated under CLIA. CLIA regulations are required to certify the validity of a test. Validity is established by measuring:
- Analytical sensitivity and specificity
- Reportable reference range or interval
For next generation sequencing tests this means several sequencing based metrics are required:
|Assessment Test||Next Generation Sequencing Specification||Sample Material|
|Accuracy||Coverage and Quality or Phred Scores||Known variants (SNP, indel) in targeted region|
|Precision||Sequence replication and coverage distribution between different operators and instruments||Reference with known variants|
|Specificity||False positive rate, degree with which a false variant is identified at a specific coverage threshold||Several samples with well characterized targets|
|Sensitivity||Likelihood test detects known variant||Several samples with well characterized targets|
|Reportable Range||Intron buffer and exon region of one or more genes||Target material with repeat regions, indels, allele drop outs|
|Reference interval||Sequence variation background measurement||Derived from an unaffected population, same as patient|
In addition to CLIA, the College of American Pathologists (CAP) has several specific guidelines for NGS labs. These include consideration for validated sample extraction, library preparation, barcoding, pooling and target enrichment. Each protocol has specific quality metrics associated with it. In addition to the wet lab, bioinformatics pipelines must be validated and tested for how precise and sensitive variants are called.
Clinical regulation of NGS based tests are undergoing rapid change as new NGS tests enter the clinic, and older ones are improved. As these changes happen, both CAP and CLIA requirements for NGS are updated on a yearly basis.
The most common NGS based assays or tests performed in a CLIA/CAP setting today include:
- Exome sequencing
- NGS gene panel sequencing
- Whole genome sequencing
- Cell free DNA sequencing
- Metagenomic sequencing
Genohub has existing relationships with 7 service providers offering nucleic acid extraction, library preparation, sequencing and data analysis under CLIA and CAP. To obtain NGS services under CLIA/CAP accreditation, submit a request here: https://genohub.com/ngs.