“I believe that NGS has the potential to become a consolidated platform for multiple genotyping approaches,” says Rajyalakshmi Luthra, Ph.D., director of the Molecular Diagnostics Laboratory at the University of Texas MD Anderson Cancer Center. “NGS has capabilities not only to identify substitution mutations and insertions/deletions (indels), but also to find fusion transcripts and copy number changes.” Instead of managing multiple diagnostic platforms, a clinical diagnostic lab may rely on just one—a next-generation sequencer.
Dr. Luthra points out that while the sequencing technology itself is already capable of detecting indels, variant-calling software packages have not yet reached desired consistency. “We continue visual inspection of raw reads until we can be sure that the software is capable of calling indels with high accuracy,” remarks Dr. Luthra.
Nevertheless, the Molecular Diagnostics Laboratory has undertaken a concerted effort to validate several NGS platforms using CLIA (Clinical Laboratory Improvement Amendments) standards. For example, the Molecular Diagnostics Laboratory clinically validated the Ion Torrent PGM (Life Technologies) using 10 ng of DNA from 70 paraffin-embedded solid tumor samples. Variants detected by sequencing 740 mutational hotspots in 46 cancer-related genes in the panel were confirmed by orthogonal conventional platforms such as Sanger sequencing and pyrosequencing.
Overall concordance between NGS and conventional platforms supported routine use of NGS in the CLIA-certified laboratory. The utility of this approach was elegantly applied to screening mutational hotspots in 54 cancer-related genes using MiSeq, Illumina’s NGS platform, as a part of diagnostic workup and treatment plan for patients with acute myeloid leukemia.
“NGS provides for a single detection platform capable of analyzing multiple genes at the same time starting with a fraction of the material required in conventional multigene analysis,” notes Dr. Luthra.
The laboratory rigorously tested several NGS-based protocols by analyzing and comparing the archival samples with known mutations and samples collected prospectively. NGS platforms showed high level of reproducibility and high detection sensitivity. This high sensitivity was useful in detecting the presence of low-level mutations that were missed by less sensitive conventional platforms during followup and relapse.