Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ballabio A, Gieselmann V (2009) Lysosomal disorders: from storage to cellular damage. Biochim Biophys Acta 1793(4):684–696. doi:10.1016/j.bbamcr.2008.12.001
Platt FM, Boland B, van der Spoel AC (2012) The cell biology of disease: lysosomal storage disorders: the cellular impact of lysosomal dysfunction. J Cell Biol 199(5):723–734. doi:10.1083/jcb.201208152
Parenti G, Andria G, Ballabio A (2015) Lysosomal storage diseases: from pathophysiology to therapy. Annu Rev Med 66:471–486. doi:10.1146/annurev-med-122313-085916
Schulz A, Kohlschutter A, Mink J, Simonati A, Williams R (2013) NCL diseases - clinical perspectives. Biochim Biophys Acta 1832(11):1801–1806. doi:10.1016/j.bbadis.2013.04.008
Aharon-Peretz J, Rosenbaum H, Gershoni-Baruch R (2004) Mutations in the glucocerebrosidase gene and Parkinson’s disease in Ashkenazi Jews. N Engl J Med 351(19):1972–1977. doi:10.1056/NEJMoa033277
Sidransky E, Nalls MA, Aasly JO, Aharon-Peretz J, Annesi G, Barbosa ER, Bar-Shira A, Berg D, Bras J, Brice A, Chen CM, Clark LN, Condroyer C, De Marco EV, Durr A, Eblan MJ, Fahn S, Farrer MJ, Fung HC, Gan-Or Z, Gasser T, Gershoni-Baruch R, Giladi N, Griffith A, Gurevich T, Januario C, Kropp P, Lang AE, Lee-Chen GJ, Lesage S, Marder K, Mata IF, Mirelman A, Mitsui J, Mizuta I, Nicoletti G, Oliveira C, Ottman R, Orr-Urtreger A, Pereira LV, Quattrone A, Rogaeva E, Rolfs A, Rosenbaum H, Rozenberg R, Samii A, Samaddar T, Schulte C, Sharma M, Singleton A, Spitz M, Tan EK, Tayebi N, Toda T, Troiano AR, Tsuji S, Wittstock M, Wolfsberg TG, Wu YR, Zabetian CP, Zhao Y, Ziegler SG (2009) Multicenter analysis of glucocerebrosidase mutations in Parkinson’s disease. N Engl J Med 361(17):1651–1661. doi:10.1056/NEJMoa0901281
Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, Gispert S, Ali Z, Del Turco D, Bentivoglio AR, Healy DG, Albanese A, Nussbaum R, Gonzalez-Maldonado R, Deller T, Salvi S, Cortelli P, Gilks WP, Latchman DS, Harvey RJ, Dallapiccola B, Auburger G, Wood NW (2004) Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304(5674):1158–1160. doi:10.1126/science.1096284
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Di Fruscio, G., Banfi, S., Nigro, V., Ballabio, A. (2017). Next-Generation Sequencing Approaches to Define the Role of the Autophagy Lysosomal Pathway in Human Disease: The Example of LysoPlex. In: Öllinger, K., Appelqvist, H. (eds) Lysosomes. Methods in Molecular Biology, vol 1594. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6934-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6934-0_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6932-6
Online ISBN: 978-1-4939-6934-0
eBook Packages: Springer Protocols