title: An effective combination of sanger and next generation sequencing in diagnostics of primary ciliary dyskinesia.
abstract: BACKGROUND
  Primary ciliary dyskinesia (PCD) is a multigenic autosomal recessive condition affecting respiratory tract and other organs where ciliary motility is required. The extent of its genetic heterogeneity is remarkable. The aim of the study was to develop a cost-effective pipeline for genetic diagnostics using a combination of Sanger and next generation sequencing (NGS).
  
  
  METHODS
  Data and samples of 33 families with 38 affected subjects with PCD diagnosed in childhood were collected over the territory of the Czech Republic. A panel of 18 PCD causative or candidate genes was implemented into an Illumina TruSeq Custom Amplicon NGS assay, and three ancestral mutations in SPAG1 were screened by conventional Sanger sequencing, which was also used for the confirmation of the NGS results and for the analysis of familial segregation.
  
  
  RESULTS
  The causative gene was DNAH5 in 11/33 (33%) probands, SPAG1 in 8/33 (24%), and DNAI1, CCDC40, LRRC6 in one family each. If the high proportion of subjects with bi-allelic ancestral mutations in SPAG1 is corroborated in other Caucasian populations, a simple Sanger sequencing test for these three mutations may serve as an effective pre-screening step, being followed by an NGS panel for other, much larger, PCD genes.
  
  
  CONCLUSIONS
  We present a combination of Sanger sequencing with an NGS panel for known and candidate PCD genes, implemented in a moderate-size national collection of patients. This strategy has proven to be cost-effective, rapid and reliable, and was able to detect the causative gene in two thirds of our PCD patients.
authors.affiliation: Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Djakow
authors.affiliation: Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Kramná
authors.affiliation: Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Dušátková
authors.affiliation: Department of Histology and Embryology, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Uhlík
authors.affiliation: Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland.
authors.forename: Juha-Pekka
authors.initials: JP
authors.lastname: Pursiheimo
authors.affiliation: Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic.
authors.forename: Tamara
authors.initials: T
authors.lastname: Svobodová
authors.affiliation: Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Pohunek
authors.affiliation: Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Cinek
mesh_terms: D000293:Adolescent; D000483:Alleles; D000954:Antigens, Surface / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D018153:Czech Republic; D005260:Female; D019204:GTP-Binding Proteins / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007223:Infant; D007619:Kartagener Syndrome / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D009154:Mutation
chemical_list: D000954:Antigens, Surface; D019204:GTP-Binding Proteins; C483942:SPAG1 protein, human
keywords: SPAG1 gene; next generation sequencing; targeted panel