The College of American Pathologists has published a checklist specific to next-generation sequencing for clinical lab accreditation. The NGS-specific checklist is part of CAP's revised molecular pathology checklist for accrediting clinical laboratories, released this week.
The checklist, available online to CAP-accredited labs, includes 18 requirements that address each step of using sequencing as a clinical test, including library creation and sample prep, the actual sequencing itself, bioinformatics, variant calling, annotation, and the final patient report.
The accreditation checklist comes a year after CAP formed a workgroup charged with developing one for next-gen sequencing (CSN 9/14/2011).
The goal of the checklist is to provide a uniform framework for validating sequencing-based clinical tests. While many clinical labs have launched next-gen sequencing tests, there has until now been no universal framework specific to NGS by which to evaluate and validate them.
While the existing molecular pathology accreditation checklist addresses Sanger sequencing and other genetic analysis technologies, it does not cover "the unique aspects and details of next-generation sequencing," Karl Voelkerding, medical director of genomics and bioinformatics at ARUP Laboratories and head of the working group that developed the checklist, told Clinical Sequencing News.
"As a working group, we determined that we needed to craft and articulate a new set of requirements, particularly because there are some aspects of next-generation sequencing that are really quite unique for clinical labs," he said.
For example, NGS allows for samples to be pooled. "This type of barcoding, pooling, sequencing, and then deconvoluting the information and assigning it back to the original sample" is an example of a technique that is not addressed by the previous checklist requirements, he noted.
Additionally, there are unique aspects regarding the analysis of NGS sequence data that were not addressed under the prior guidelines.
The new guidelines are effective immediately, so any clinical lab that wants to start offering NGS-based diagnostic tests in a CAP-accredited lab will have to adhere to these requirements . Laboratories that are already accredited by CAP will need to make sure their NGS-based tests adhere by their next inspection, Voelkerding said.
Once accredited, clinical labs are subject to inspection every two years by CAP and in the off-years are required to do their own self-inspection. Thus, labs will likely be evaluated based on the new checklist starting this fall, Voelkerding said.
While this may seem like a quick turnaround, Voelkerding said that labs that are already offering NGS-based tests under CLIA are likely following many of the basic principles described in CAP's NGS-specific requirements.
"The CLIA guidelines have many of the same or similar guiding principles in them, for example, the need to establish the sensitivity and specificity of a diagnostic assay," he said, but were developed "well before the advent of next-gen sequencing." The CAP checklist requirements "add another layer of detail" specific to the features of NGS.
Among the requirements in the new guidelines are validated sample-prep protocols, including library creation; any barcoding, indexing, and pooling steps; target capture, enrichment, and amplification. Quality scores for each base must be taken into account when determining accuracy of a test, and laboratories must document expected performance characteristics according to the manufacturer as well as measured performance characteristics.
Additionally, all steps for introducing new reagents or analytical methods must be validated.
Laboratories must also have a policy for performing confirmatory testing and must maintain an ongoing record of the sensitivity and specificity of tests, as well as their false positive and false negative rates.
The laboratory must also have a system for interpretation and reporting of sequence variants, including a policy regarding reporting clinically significant "incidental" or unanticipated findings.
These protocols must have quality metrics attached to them, and like the process for generating sequence data, if any upgrades or changes are introduced, the process must be re-validated.
Similarly, the bioinformatics pipeline for alignment and variant calling must be documented with quality metrics such as sensitivity, specificity, false positives and negatives, reproducibility, and precision for the types of variants assayed — for instance single nucleotide variants, insertions and deletions, and homopolymers or repetitive sequences.
The checklist also includes requirements for data storage and confidentiality in order to ensure a laboratory is consistent in the types of data it stores and for how long.
Finally, all the methods, systems, reagents, and bioinformatics pipelines used in the clinical test must be traceable through the final patient report.
Voelkerding said that one of the challenges in developing the requirements is that labs use different sequencing platforms, sample-prep reagents, and bioinformatics tools. "So, we needed to articulate a series of checklist requirements that address the fact that laboratories are using different platforms and that those platforms continue to evolve at a fairly quick pace."
However, he said, the group wanted the checklist to represent "guiding principles of good lab diagnostic practices," meaning the requirements would have to stand the test of time at least for the next several years — a difficult feat in a field that moves so quickly, he said.
Voelkerding said that the working group will continue to evaluate other options to include in the requirements. For instance, he said that the group has been discussing the option of creating separate requirements for oncology and infectious disease tests.
These additions would not be an extensive number of new requirements, he said. Rather the requirements would seek to address features that are unique to those types of tests, namely sample heterogeneity. In an oncology sample there will be a mixture of normal DNA and tumor DNA, he said. Similarly, an infectious disease sample would likely include both human DNA and pathogen DNA.
There are already CAP checklist requirements specific to oncology and infectious disease tests, so a future update may be as simple as indicating that those checklists would apply to an NGS test for oncology or infectious disease, he said.
Additionally, CAP requirements can be introduced or updated on a yearly basis, so over the next year as clinical labs go through the CAP accreditation process, the group will solicit feedback from both the labs as well as the inspectors, to identify any requirements that need to be revisited. Often it's just a matter of revising the language to clarify the intent of the requirement, Voelkerding said.