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Next-Generation Sequencing Approaches to Define the Role of the Autophagy Lysosomal Pathway in Human Disease: The Example of LysoPlex.
Next-Generation Sequencing (NGS) technologies have deeply changed the throughput of genetic testing allowing analyzing millions of DNA fragments in parallel. One key application is the understanding of genetically heterogeneous and complex diseases where 50-100 different genes may converge to control the same pathways. These disorders cannot be studied using traditional approaches, based on gene-by-gene Sanger sequencing. We have set up an NGS protocol based on a specific selection of DNA regions belonging to about 900 genes of the autophagy-lysosomal (ALP) pathway. We here specify all the technical steps and challenges of our protocol, named LysoPlex. This is based on the Haloplex technology and together with high-coverage sequencing empowers a high and uniform coverage of ALP genes. LysoPlex outplays other NGS applications in sensitivity and specificity, providing an accurate picture of all variations in ALP genes.
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