Most genetic tests rely on determining the sequence of nucleotides in a gene. For the past 35 years, the “gold standard” for gene sequencing has been the Sanger method, in which the sequence is determined for one region of DNA at a time. But in recent years, so-called “next generation sequencing” (NGS) has provided an alternative method. As technology improves, and the number of potential genes of interest grows, NGS is increasingly being offered by gene testing laboratories for many applications. But not all NGS platforms are the same. Because of its experience, expertise, and stringent quality control, The Athena Diagnostics NGS platform provides superior results across the genome.
NGS is also called “massively parallel sequencing,” because the technique analyzes the sequence of multiple sections of DNA at the same time. That can be an advantage for large gene panels, used when there is no single clear gene of interest. That is increasingly the case in disorders such as epilepsy, for which the number of genes is growing virtually every year. Sample preparation is more laborious than with the Sanger method, “but you get gigabases of information out of a run,” says Corey Braastad, PhD, Scientific Director for Athena Diagnostics.
That extra up-front work makes the difference in the quality of the sample, and therefore the quality of the genetic information that emerges. One critical step is in sample enrichment. Many testing labs use polymerase chain reaction (PCR) to amplify the target sequence. But PCR-based amplification, Braastad notes, runs the risk of “allele drop-out,” when an initial target mismatch leads to loss of the target as the rest of the DNA is amplified around it. The alternative, used at Athena Diagnostics, is hybridization, in which short oligonucleotides, customized for the target sequence, act as bait to fish out the target from the sample. “Hybridization is much less susceptible to drop-out,” Braastad says.
The second critical feature of NGS is “coverage,” referring to the number of times a specific piece of DNA is sequenced during a run. Sequencing the same segment repeatedly increases accuracy. Many labs use average coverage as their quality standard, without noting minimum coverage. “But it is quite possible to have a high average coverage while still missing some regions entirely,” Braastad points out. That is why Athena Diagnostics uses a minimum coverage of 20X, as well as an average coverage of 30X, as its quality standard.
Athena Diagnostics has extensively tested its NGS platform against Sanger sequencing, and found complete concordance of results, with zero false positives and zero false negatives. Most labs have not performed the same level of concordance analysis, because they do not have the same depth of clinical material with which to make these comparisons. Those labs that have published the results of their concordance analysis do not approach the level of concordance that Athena Diagnostics has achieved.
“Technical accuracy is expected in a CLIA-certified lab,” Braastad says, but not every lab attains the same level of accuracy with its NGS platform. “ I believe we lead the pack on that score.”