Head of Next Generation Sequencing Facility, TIGEM
Professor of Medical Genetics, Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Italy
Next Generation Sequencing Facility
High-throughput sequencing technologies have revolutionized all fields of biosciences and are essential to the discovery of gene functions and their involvement in disease conditions. Next generation sequencing (NGS) produces a huge volume of sequencing data with cheap costs/base and is applied to both functional genomics (e.g. global analysis of gene expression) and structural genomics (e.g. genomic sequencing of individual genomes). Our facility uses SBS Illumina HiSeq technology.
The NGS facility, coordinated by Vincenzo Nigro, has performed in-house whole-exome sequencing (WES), RNA sequencing and preferential exome sequencing. Since 2012, the NGS facility has analyzed 2,460 samples, of which about 440 were analyzed by WES, and the remaining by targeted NGS approaches. New disease genes, such as LGMD1F, have so far been identified.
The main instruments used are the Illumina HiSeq1000 system and the computing cluster.
Ferraro MB, Savarese M, Di Fruscio G, Nigro V, Guarracino MR (2014). Prediction of rare single-nucleotide causative mutations for muscular diseases in pooled next-generation sequencing experiments. J Comput Biol. 21(9):665-75. doi: 10.1089/cmb.2014.0037.
Torella A, Fanin M, Mutarelli M, Peterle E, Del Vecchio Blanco F, Rispoli R, Savarese M, Garofalo A, Piluso G, Morandi L, Ricci G, Siciliano G, Angelini C, Nigro V (2013). Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F. PLoSOne. 8(5):e63536. doi: 10.1371/journal.pone.0063536.
Rotundo IL, Lancioni A, Savarese M, D'Orsi L, Iacomino M, Nigro G, Piluso G, Auricchio A, Nigro V (2013). Use of a lower dosage liver-detargeted AAV vector to prevent hamster muscular dystrophy. Hum Gene Ther. 24(4):424-30. doi: 10.1089/hum.2012.121.
Lancioni A, Rotundo IL, Kobayashi YM, D'Orsi L, Aurino S, Nigro G, Piluso G, Acampora D, Cacciottolo M, Campbell KP, Nigro V (2011). Combined deficiency of alpha and epsilon sarcoglycan disrupts the cardiac dystrophin complex. Hum Mol Genet. 20(23):4644-54. doi: 10.1093/hmg/ddr398.
Rotundo IL, Faraso S, De Leonibus E, Nigro G, Vitiello C, Lancioni A, Di Napoli D, Castaldo S, Russo V, Russo F, Piluso G, Auricchio A, Nigro V (2011). Worsening of cardiomyopathy using deflazacort in an animal model rescued by gene therapy. PLoSOne. 6(9):e24729. doi: 10.1371/journal.pone.0024729.
The current objective of Nigro’s team is to identify novel mechanisms of disease in undiagnosed myopathic patients with the help of next generation sequencing. We identified more genetic variations in the human muscle genes than we initially expected to find. This suggests that currently used tests may exclude some very relevant information. We have created various panels of genes involved in known and potentially new forms of muscle disorders. We have already studied about 750 unresolved cases of myopathies, excluding their obvious candidate genes We have identified known pathogenic variants in about 40% of the patients and discovered that at least 20% patients show multiple true disease-associated variants that suggest complex inheritance. Moreover, we frequently detected rare variants of unknown significance in the largest muscle genes in addition to variants in new genes. This effort, combined with whole exome sequencing and RNA sequencing will shed light on new forms of muscular dystrophies and eventually allow us to diagnose patients.