UPDATE January 2020:
Just in time for the 4th of July, on July 3rd 2019, we installed and activated an Illumina iSeq-100 DNA sequencing system. Dr. Mitton, Wendy Dailey, Jennifer Felisky, Naomi Haque, Michael Sun, and Ed Guzman from the ERI are all contributing to the setup and eventual ongoing use of this system for high-throughput DNA-sequencing to discover mutations in 8 genes involved in the following rare and inherited conditions:
FEVR (Familial Exudative VitreoRetinopathy), Norrie Disease, and Retinoschisis (RS).
This was done to establish the first DNA-sequencing research service at Oakland University applied to the discovery of genetic disease causing mutations. Our goal is to use these molecular investigations to advance our clinical understanding of these blinding diseases that impacting patients of Associated Retinal Consultants, (Royal Oak Michigan).
Unlike several health care systems in Europe and Canada, DNA sequencing costs are not covered by health insurances in the United States. So for Americans, the access to even DNA-sequencing of genes we already know may be involved in a patient’s disease is simply not available because of the prohibitive costs. As molecular biologists working in vision science, my lab group has developed and then tested a targeted DNA-sequencing panel to analyze many genes related to these rare conditions for only a few hundred dollars per sample. Several developments in technology over the last several years have now made this possible.
- We use a strategy called “Ampliseq “which is based on PCR (Polymerase Chain Reaction). Using a computational (in silico) design process from Illumina, we designed a large number of PCR primer pairs to copy and amplify all the exon and flanking intronic DNA sequences from 8 genes of interest. These are genes that we either know or think can be involved in these rare pediatric retinal diseases.
- Each PCR product is about 250 base pairs in length and the total sequence from these 8 genes is over 30,000 base pairs. The primer pairs are mixed in three different pooled combinations (three PCR reactions per patient).
- Up to 16 patient DNA samples are processed at the same time by PCR amplification at one time. The PCR products are linked to Illumina end sequences, purified, QC tested to make what is called a sequencing library. These are stored frozen until about 32 to 40 samples are ready.
- The sequencing libraries from, say, 40 patients are mixed together and loaded into a processing cartridge that is then loaded into the iSeq100. The single use cartridge contains all the sequencing microfluidic chemistry reagents, and a sequencing flow cell is also plugged into the cartridge.
- Using a fluorescent diod-array imaging system, 4.5-Million PCR library products, averaging 250 base pairs each in length, are sequenced simultaneously to generate well over 1.2 GigaBases of DNA sequence. The illumina primers contain a unique 4-basepair sequence for each patient’s sequencing library, so the iSeq100 software then sorts all the sequencing reads into separate folders for each patient using this “DNA barcode”.
- Each Patient’s DNA sequence fragments are then aligned to a reference Human genome sequence and then any differences in the patient’s DNA sequence from the reference genome is reported to us.
- We use special bioinformatics software in the cloud and some programming tools called “R” to sort these DNA sequence variants and discover changes that would change or impact the normal proteins encoded by the 8 genes of interest.
To start in late 2019, I am happy to report here that we sequenced the first 35 patients, most who have FEVR. This inherited condition causes the lack of a full and normal retinal vasculature, especially in the periphery of the retina, resulting in a lack of oxygen to the cells of the neural retina. FEVR, Norrie Disease, and Retinoschisis can all have effects that lead to blindness.
The result of the above process is that we now have the ability to test 8 genes for less than $300, and complete the testing in one week starting from a few drops of blood. Previously, only 2 to 3 of these genes in a DNA sample could be sequenced for about $4,500, using different service laboratories, taking months. Not only will this make it possible to sequence more genes for more patients, we can also use this new genetic testing research system to provide advanced training to Oakland University’s undergraduate, graduate and medical school students.
The next batch of patient tests samples, the first in 2020 will be sequenced this month, February 2020 and we will continue to sequence more and more patients to help us find the underlying causes of these rare genetic blinding diseases in children. Welcome to 2020.