To evaluate the newly developed next generation sequencing assay by analyzing patients with isolated eye abnormalities collected in earlier studies including patients enrolled through eyeGENE program: This project began several years ago in the OGFVB/NEI. We developed a screening tool by the resequencing CHIP on 93 genes provided an opportunity for these samples from patients enrolled in NEI clinical research for years. Our manuscript was published in the IVOS later in 2011. Later on, we adapted the next generation sequencing (NGS) technology. We developed a new panel composed of 184 genes related to retinal function and development. By NGS and microdroplet PCR technology, we have been more actively analyzing patient with variety categories of retinal dystrophies in a more efficient and accurate approach. We hired a company, RainDance Technologies, Inc., in designing a primer library for Retinal Dystrophy Panel (RD panel). Using the developed primer library, RainDance Technologies, Inc. we have performed NGS sequencing for more than 275 samples with a variety of ophthalmic diseases. Those samples have been sequenced in our newly purchased MiSeq next generation Sequencer from Illumina. We are continuing the procedures to analyze more patients with RP. Meanwhile, we have built our own in house bioinformatics pipeline to analyze NGS data. We are capable to analyze all kinds of NGS data, including WES data. Using this pipeline, we have analyzed NGS data from different projects. Beforehand, our Next Generation sequencing data was outsourced. In order to comply with the new direction of the DNA Diagnostics Laboratory, we purchased a package of software for NGS data process pipeline for processing and analyzing all of our next generation sequencing data. To date, we have analyzed over 173 samples for several projects including the following: o Perform analysis for NISC project. Assisted in the development of the pipeline of 100 samples to be analyzed from NISC. o Perform analysis for whole exome sequencing project (WES). Analyzed samples and developed protocol for whole exome sequencing analysis. We also used these samples to search for ACMG common genes. We have also purchased a software package for mutation analysis, Alamut. This software assists us in the determination of variant pathogenicity. Alamut consolidates data from 3 mutation algorithms, population frequencies, nucleotide and amino acid conservation, protein structures, and splicing information into one platform. This saves the DDL lab significant time and effort when analyzing variants. New approach to perform NGS sequencing We are currently testing a new method provided by Agilent Technologies that will allow us to provide a superior method for comprehensive and efficient targeted sequencing that offers cost efficiencies and a streamlined workflow; while, accelerating the turn-around time from sample to date and providing deep coverage of genomic regions of interest. This project will allow us to potentially decrease the turnaround time frame <2 weeks /16 samples along with the cost per sample to $284. Clinical laboratory for FDA approved clinical trial, XLRS2 project NEI started a FDA approved clinical trial to study the gene therapy for X-linked Retinaoschisis using RS1 gene ocular transfer technology. We are the clinical laboratory for post-therapy evaluation of the XLRS2 clinical trial. We have processed 36 Serum samples, 7 AAV Vector Dilution Storage and 3 site visits in support of XLRS2 clinical trial. We have performed 36 serum extractions on seven participants, stored 7 AAV vector dilutions and had three site visits. Time spent processing each serum sample is approximately 45 minutes to 1 hour. We created a custom DDL Variant Report to use with Alamut for variant analysis. The report provides easy access to variant information without the need for special software.
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