Identification and Characterization of Splicing Defects by Single-Molecule Real-Time Sequencing Technology (PacBio)

Article type: Research Article

Authors: Savarese, Marco^{a; b; 1; *} | Qureshi, Talha^{a; b; 1} | Torella, Annalaura^{c; d} | Laine, Pia^e | Giugliano, Teresa^c | Jonson, Per Harald^{a; b} | Johari, Mridul^{a; b} | Paulin, Lars^e | Piluso, Giulio^c | Auvinen, Petri^e | Nigro, Vincenzo^{c; d} | Udd, Bjarne^{a; b; f} | Hackman, Peter^{a; b}

Affiliations: [a] Folkhälsan Research Center, Helsinki, Finland | [b] Department of Medical Genetics, Medicum, University of Helsinki, Helsinki, Finland | [c] Dipartimento di Medicina di Precisione, Università degli Studi della Campania “Luigi Vanvitelli”, Naples, Italy | [d] Telethon Institute of Genetics and Medicine, Pozzuoli, Italy | [e] Institute of Biotechnology, University of Helsinki, Helsinki, Finland | [f] Vaasa Central Hospital, Vaasa, Finland

Correspondence: [*] Correspondence to: Marco Savarese, Ph.D., Folkhälsan Institute of Genetics, Department of Medical Genetics, University of Helsinki, Biomedicum, Haartmaninkatu 8 -Pb 63 00014 Helsinki, Finland. Tel.: +358 294125069; E-mail: marco.savarese@helsinki.fi.

Note: [1] These authors contributed equally to the work.

Abstract: Although DNA-sequencing is the most effective procedure to achieve a molecular diagnosis in genetic diseases, complementary RNA analyses are often required. Reverse-Transcription polymerase chain reaction (RT-PCR) is still a valuable option when the clinical phenotype and/or available DNA-test results address the diagnosis toward a gene of interest or when the splicing effect of a single variant needs to be assessed. We use Single-Molecule Real-Time sequencing to detect and characterize splicing defects and single nucleotide variants in well-known disease genes (DMD, NF1, TTN). After proper optimization, the procedure could be used in the diagnostic setting, simplifying the workflow of cDNA analysis.

Keywords: Long-read sequencing, single molecule real time, PacBio, splicing

DOI: 10.3233/JND-200523

Journal: Journal of Neuromuscular Diseases, vol. 7, no. 4, pp. 477-481, 2020