DNA Sequencing for Families with Rare Inherited Retinal Diseases.

Meet Pearl. The new DNA-sequencer in PRRL (Pediatric Retinal Research Laboratory)


Just in time for the 4th of July, on July 3rd, 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 will establish the first sequencing research service at Oakland University applied to the discovery of genetic diseases. 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 and so we have developed and then tested a targeted 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 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, a few hundred PCR primer pairs were designed to copy and amplify all the exon and flanking intron sequences from 8 genes of interest.
  • Each PCR product is about 250 base pairs in length and the total sequence from these 8 genes is about 27,000 base pairs. A few hundred primer pairs are mixed in three different pooled combinations (three PCR reactions per patient).
  • Up to 16 patient DNA samples are processed 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 36 to 45 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 microscope imaging system, 4.5-Million PCR library products, averaging 250 base pairs each in length, are sequenced simultaneously to generate a total of 1.2 GigaBases of DNA sequence. The illumina primers contain a unique 4-basepair sequence for each sample, so the iSeq100 then sorts all the sequencing reads into separate folders for each sample (Patient).
  • Each Patient’s DNA sequence fragments are aligned to the reference, known, Human genome sequence and then any differences in the Patient’s DNA sequence is determined.
  • We use special bioinformatics software developed at Oxford University (UK) to sort these DNA sequence variants and discover changes that would change or impact the normal proteins encoded by the 8 genes of interest.

The result of the above process is that the current ability to test 2 to 3 of these genes in a DNA sample for about $4,500, using different service laboratories, will now be 8 genes for less than $400 of cost. Not only will this make it possible to sequence more genes for more patients, Dr. Mitton is using this new genetic testing research system to provide advanced training to Oakland University’s undergraduate, graduate and medical school students.