NGS and diagnosis

The molecular diagnosis of monogenic forms of diabetes and obesity is crucial as the identification of a genetic mutation in a specific gene is likely to improve quality of life or even to increase patients’ life expectency (along with a more personalized treatment). Furthermore, although monogenic diabetes is not common (2% of diabetics), it has been demonstrated that this molecular diagnosis was cost-effective in the USA. 

Before 2010, this molecular diagnosis was uniquely based on Sanger sequencing. Even if this method is very sensitive and reliable, its throughput is very low and the method is very expensive and labor-intensive. For instance, the sequencing of three genes (ABCC8, KCNJ11 and INS) involved in neonatal diabetes cost 2,800€ the French health system.

We have developed a method enabling a reliable, fast, easy and inexpensive (< 300€) sequencing of all genes involved in monogenic forms of diabetes and obesity. This method is based on PCR in lipid microdroplets (RainDance technology, enabling a very high throughput as a vial can include millions of droplets; please see figure below), in combination with Illumina NGS (for instance using MiSeq as the target is rather small).

Furthermore, we sought to assess other accessible protocols in order to improve the genetic diagnosis in terms of cost, reliability, sensitivity, and speed, we have assessed three other technologies in addition to RainDance: Agilent SureSelect, Agilent HaloPlex and Illumina Nextera. Of note, for the preparation of both RainDance and SureSelect libraries, DNA is sonicated while DNA is digested with enzymes for the preparation of both Nextera and HaloPlex libraries. We have observed that targets were well covered via SureSelect, RainDance and HaloPlex; while the coverage was quite poor via Nextera. More importantly, we have observed that both Nextera and HaloPlex technologies lead to a high rate of false-positive and false-negative mutations (please see figure).

Therefore, from our unbiased comparative study, we recommend Agilent SureSelect and RainDance for the molecular diagnosis of monogenic disorders.