With the rapid advancement of high-throughput next-generation sequencing (NGS) technologies, the cost of sequencing continues to drop and the platform is migrating from genome centers to core facilities to individual clinical laboratories. Greater numbers of clinical molecular diagnostic laboratories have begun implementation of their own laboratory-developed tests (LDTs) using NGS platforms. But is NGS a molecular method ready for prime time in diagnostic labs?

Many labs may have a few questions in common when considering adding NGS to their offerings: What is NGS? Is it expensive? I feel like I am behind the curve and need to get up to speed; what factors do I need to consider as I talk to my hospital administrator about adding this technology? Will my medical staff want this testing?

General themes that will help pave the way for laboratorians looking to answer the above questions are explored.

Instrumentation
After the first wave of ultra-high-throughput NGS instruments, manufacturers attempted to democratize sequencing by marketing relatively low-throughput benchtop units. Roche, Ion Torrent of Life Technologies and Illumina launched 454 Junior, PGM and MiSeq, respectively. Benchtop sequencers are attractive and practical for individual laboratories, as the capital investment and cost of each run is much more affordable. Many labs may wish to detect mutations/sequence changes of their favorite genes for smaller runs and the low-throughput instruments are more amenable.

However, data analysis is a significant hurdle for routine use of NGS in clinical labs. Typically, full-time bioinformatic support is required when using ultra-high-throughput NGS sequencers. Vendors have realized this challenge, and are striving to ease the implementation in clinical labs. Currently, benchtop sequencers include software for primary, secondary and some tertiary analysis. Still, instrument vendors typically do not bundle annotation capabilities with the instrument and extra resources are needed to set up a customized analysis pipeline to generate fully annotated variant call reports.

With fierce competition in the NGS field, manufacturers improve their consumables, and sometimes instruments, almost quarterly to maintain a competitive advantage in the marketplace. This creates a challenge for clinical labs; manufacturers realize that and have begun offering options for labs to continue to use the consumables they have validated. In addition, they are working on FDA-cleared benchtop sequencers for diagnostic purpose. This is all welcome news for labs.

CLIA Laboratories
Obviously, NGS is not going to be the perfect fit for all the tests in a CLIA-regulated laboratory. NGS for a low-volume test with fewer mutations/sequence changes is not as efficient as traditional molecular detection methods. However, the capability of NGS instruments makes whole genome sequencing or screening large panels of mutations/genes possible when the results can support patient care.

Before the advent of benchtop sequencers, a number of academic and commercial CLIA service labs have brought up tests on ultra-high-throughput NGS sequencers. Such tests are financially sensible only when the test volume is high, or the number of genes/mutations on each run is large. For most CLIA service labs, the initial capital investment and throughput capacity of the ultra-high-throughput NGS sequencers are not optimally compatible with a clinical laboratory operation. The introduction of lower-throughput benchtop NGS sequencers makes it easier to justify the acquisition of an NGS platform and manage the cost of each run.

Despite efforts to streamline workflow, the technology is still complex compared to other common technologies in MDx labs. NGS is not yet a "plug and play" instrument. It takes two major steps, library preparation and massive parallel sequencing. While the sequencing portion is established and requires minimal optimization, library preparation can be done via many technologies, as long as the resulting sample libraries fit into the sequencing workflow. Even if each NGS instrument manufacturer had its own library preparation products, which would be a good starting point, there is an opportunity to customize the workflow in each lab. Not surprisingly, there are a number of commercial solutions tailored to library preparation. For labs new to the technology, there is much upfront investigation required to chart the optimal NGS path for one's own lab's characteristics.

In addition, there is no standard protocol on how to launch an NGS test, though useful articles are appearing monthly.^{1, 2} Validation and verification are based on the discretion of the laboratory director now. Whether to perform confirmatory testing of positive results or report patient results based on NGS alone is one such point of consideration. The latter option would require strict and clever controls with stringent cut-off criteria to identify mutations. Dealing with variants of unknown clinical significance is also an issue worth serious consideration. Labs should look at their educational resources and the medical staffs they support as additional variables in adding NGS testing.

Bioinformatic expertise is also necessary to establish an automated and streamlined analysis workflow for each newly developed NGS diagnostic test, even with benchtop sequencers that generate less data. Such a requirement is warranted as labs continue to either introduce new tests or improve existing sequencing based tests. As most CLIA labs do not have dedicated bioinformaticians, requirements from both "wet lab" and in silico analysis during the implementation of NGS technology will lead to notable changes on how a CLIA lab functions in the future.

The topic of reimbursement, always ­challenging, might be even more so for NGS because the status quo for gene and mutation analysis is well recognized by payers. Will they properly reimburse the NGS tests? How will the new CPT codes affect the financial proposition of NGS in the CLIA laboratory? Reimbursement is a variable at this time. Some institutions may wish to underwrite to remain cutting edge. This part of the equation is similar to the earliest days of molecular diagnostics when questions of reimbursement versus being ahead of the curve were considered by early adopters. As more CLIA labs are venturing into NGS technologies, government agencies, regulatory and professional organizations are working to establish regulations and guidelines on the use of NGS methods for patient care.

1. Gargis A, Kalman L, Berry M, et al. Next-generation sequencing: Guidance for the translation from research to clinical applications. Nat Biotechnol. In Press.
2. 2. Schrijver I, Aziz N, Farkas DH, et al. Opportunities and challenges associated with clinical diagnostic genome sequencing: A report of the Association for Molecular Pathology. J Molec Diagn. 2012 Aug 20 [Epub ahead of print].