Introduction to Next Generation Sequencing (NGS)

In Week 3 we introduced you to the traditional techniques used for genetic testing: PCR and Sanger sequencing. These techniques allow the analysis of around 500 nucleotides of DNA a time. For many genetic disorders, including diabetes, there are many genes that, when mutated, can cause the disease. For example, neonatal diabetes can be caused by mutations in over 20 genes. Sequencing all of them, a piece at the time, for every patient would be simply impossible. What to do then?

Now a new way to analyse genes is available: next-generation sequencing. This uses a new approach that allows sequencing of many genes at the same time. This is known as massively parallel sequencing. The main applications of this technique in diagnostics are:

• Custom-designed panel of genes (i.e. all the genes that cause monogenic diabetes)
• Sequencing of all the coding regions (the exome) in the human DNA: Whole Exome Sequencing
• Sequencing of the entire human genome: Whole Genome Sequencing

You will hear a bit more about the technical details of next generation sequencing in the next sections. But first let’s consider how these techniques can help in finding a genetic diagnosis in patients with monogenic diabetes.

A gene panel for monogenic diabetes

There are currently 22 genes in which variants are known to cause neonatal diabetes and 16 with mutations causing MODY. We have designed a custom next-generation sequencing panel that includes all these genes and we are now routinely testing patients with a suspected diagnosis of monogenic diabetes using this technique. You can find more information about this test on our Diabetes Genes website.

The possibility of testing all the disease-causing genes has a huge impact on the patients’ clinical management. Let’s take neonatal diabetes as an example. Each of the 22 genes defines a different subtype of neonatal diabetes. Each subtype has different treatment options, is associated with the development of various additional features and has a different prognosis. Back when we undertook genetic testing by analysing one gene at the time, we needed to decide which gene we thought was most likely to be the cause of the disease. The decision was guided by the clinical features of the patient at the time of referral. If we didn’t identify a mutation in the gene we had chosen, than we were stuck until the patient developed additional complications that were consistent with one of the neonatal diabetes subtypes. At this point the genetic testing was just confirming a diagnosis made on the basis of the clinical features.

Now, we don’t need to try to guess the gene anymore, we can use next generation sequencing to test all the neonatal diabetes genes as soon as the patient is referred for genetic testing. This means that for an increasing number of patients the genetic diagnosis comes before the development of the additional complications. This way the genetic result informs clinicians on optimal treatment, on disease progression and raises awareness on the future development of additional features. In other words, the genetic result is now making the diagnosis rather than just confirming it.

_{From The effect of early, comprehensive genomic testing on clinical care in neonatal diabetes: an international cohort study, De Franco et al, Lancet. 2015 Sep 5;386(9997):957-63. Reproduced under a Creative Commons (CC by 4.0) licence.}

If you would like to know more about the targeted test for all MODY genes we suggest you look at this material:

• Improved genetic testing for monogenic diabetes using targeted next-generation sequencing, Ellard S et al 2013, Diabetologia

• The effect of early, comprehensive genomic testing on clinical care in neonatal diabetes: an international cohort study, De Franco E et al 2015, Lancet. This Lancet publication describes how genomic testing changes the management of neonatal diabetes.