Smart Sequencing Through the SmartChip: A Tool To Replace Conventional PCR-based Target Enrichment

Abstract

Purpose: To study rare eye diseases using next generation sequencing (NGS) in a diagnostic setting, the biggest challenge is to obtain a complete and uniform coverage. PCR based target enrichment offers complete coverage due to its robustness, high specificity and sensitivity. However, PCR set-up can be time consuming. To overcome this challenge, our CLIA-certified molecular diagnostic lab has replaced a conventional PCR based target enrichment method with the SmartChip TE System by WaferGen Biosystems.

Methods: A SmartChip TE DNA Panel allows for thousands of singleplex PCR reactions to be processed in a simple setup that is less error prone than conventional methods. After PCR, the enriched targets from the individual wells are pooled together and sequenced by NGS. A Stargardt/Macular Dystrophy panel printed with 549 unique essays covering the entire coding regions from 10 genes has been developed. The region is covered in duplicate with two unique primer sets to avoid random PCR failures and allelic drop out. Library preparation following target enrichment allows for the entire workflow to be completed in a single day.

Results: The Stargardt/Macular dystrophy panel has been validated with minimum coverage of >100X and an average depth of >500X. A 126 Stargardt patient cohort has been analyzed: 84 patients tested positive; six patients had a single mutation identified in the ABCA4 gene, and 36 patients tested negative. Deep intronic mutations in ABCA4, reported in two separate publications from 2013 and 2014, respectively, were efficiently analyzed using the SmartChip Panel for all patients in the cohort.

Conclusions: WaferGen’s innovative technique is not only more efficient and cost effective but also allows for accuracy of detection to reach higher levels. SmartChip TE is a highly smart and cost effective platform compared to other PCR enrichment methods. With an incidence rate of approximately 1 in 8000 for Stargardt disease, we are able to offer affordable testing for patients and families. Additionally, the ease of adding new primers to improve coverage and new sequencing regions and new genes further streamlines the mutation detection pipeline. Panels for pigmentation/albinism and retinal dystrophy have also been developed on the WaferGen SmartChip TE platform. Taken together, we have developed an ideal solution of testing targeted gene panels for CLIA certified laboratories.