Rapid development of clinical and diagnostic applications for NGS benchtop sequencers

Besides research projects, there is growing interest in applying NGS technologies to diagnosis and clinical pratice. The new benchtop sequencers have dramatically reduced the costs, making also the analysis of a reduced number of samples cost effective and allowing small center to jump in the NGS field. While the big instruments (like HiSeq 2000 and 2500 or the recently released Ion Proton) remain intended for large sequencing centers or whole genome/exome complex analysis, the small ones (MiSeq and Ion Torrent) are being shifted markedly toward dignostic applications. The final aim for both Illumina and Life Tech is to obtain clinical certification for target resequecing protocols on their machine, even if this goal still seems not at hand in the short period. Indeed, the error and false positive rates of NGS platforms, togheter with lack of standards in the analysis pipeline, remain major concerns and prevent NGS technology from a rapid access to the clinical field.

However, the use of target resequencing and WES has demonstrated to be a powerful tool in molecular diagnosis and mutation discovery. Several papers have been published reporting the identification of new mutations for known syndromes as well as for new rare diseases. Some interesting examples are: - - the use of WES to identify molecular defects in Usher syndrome; (SOLiD, GAIIx and 454 GS FLX)
- the application of 454 technology for the dignosis of inherited retinal degeneration;
- a novel mutation identified in CUL7 gene in a patient with an undiagnosed growth disorder (HiSeq 2000);
- application of WES in a consaguineous family that lead to the identification of mutations in GTDC2 gene as cause of Walker-Warburg Syndrome (HiSeq 2000);
- identification of KCNQ2 defects as cause of Ohtahra syndrome, a kind of neonatal epileptic encephalopathy (HiSeq 2000);
- identification of the molecular defect in the calcium channel gene CACNA1S in a children with atypical episodic muscle weakness (HiSeq 2000);
- discover of compound heterozygous mutations in the PKHD1 gene as cause of recessive polycystic kydney disease (HiSeq 2000);
- identification of missense mutations in SLC29A3 gene as cause of Dysisteosclerosis (HiSeq 2000), a peculiar form of ostepetrosis;
- study on a large family affected by essential tremor neudegenerative syndrome that revealed defects in the FUS gene (SOLiD);
 - discovery of ANK3 mutations as associated with autism syndrome applying WES to a cohort of about 60 individuals (GAIIx).
These publications indicate that Illumina HiSeq sequencers dominate the NGS arena by now. We are curious to see if something is going to change with the capillary installation of Ion Proton sequencers.

A case worth mentioning is for sure cystic fibrosis, a disease caused by impairment of the CFTR gene with more than 1000 mutations identified so far. Even if a major percentage of cases are due to a restricted set of about 20 mutations, the possibility to quickly sequence the entire gene would simplify and speed-up the molecular diagnosis, particularly for ethnic groups with a high prevalence of rare mutations. In the past year some paper came out describing the application of Ion Torrent PGM sequencer for a quick and rapid detection of CFTR mutations and the set-up of a diagnostic protocol based on this NGS platform (see a recent example here). Recently, the University of Medicine and Dentistry of New Jersey's, Institute for Genomic Medicine (IGM), has announced that this procedure has been adopted as the standard diagnostic test for Cystic Fibrosis. A multiplexing protocol on a 316 chips allows to lower the cost of the analysis of a DNA samples at $200. This analysis looks at the 1,000 most relevant mutations identified so far providing an 98% detection rate, a remarkable improvement over the 88% obtained with the previous protocol. IGM also offers a complete mitochondrial DNA analysis for about $500 and revealed the intention to expand the NGS based diagnostic protocols to other frequently asked tests.

The diagnostic applications also represent a top priority for the NGS leader companies. Illumina has recently announced its new TruSight kits for the MiSeq (see also a post on GenomeWeb) which are targeted to various genetic disease such as autism, cardiomyopathies and pediatric inherited diseases. To complete the offer they also include a sort of Exome kit that covers all the genes reported in the Human Gene Mutation Database. Four months ago Life Technologies has presented its AmpliSeq kit on Inherited disease and with the AmpliSeq Designer they provide a tool for customer tailored target resequencing. Both companies also sell solutions targeted to the analysis of cancer genes, which is another diagnostic area with a tremendous expansion.

For sure NGS has enormously boosted the development of sequencing applications for diagnostic testings as well as the discovery of novel functional variants, opening the path leading to the long waited Personal Genomics and Personal Medicine.