title: Evaluating the Calling Performance of a Rare Disease NGS Panel for Single Nucleotide and Copy Number Variants.
abstract: Variant detection protocols for clinical next-generation sequencing (NGS) need application-specific optimization. Our aim was to analyze the performance of single nucleotide variant (SNV) and copy number (CNV) detection programs on an NGS panel for a rare disease.
  
  
  
  Thirty genes were sequenced in 83 patients with hereditary spastic paraplegia. The variant calls obtained with LifeScope, GATK UnifiedGenotyper and GATK HaplotypeCaller were compared with Sanger sequencing. The calling efficiency was evaluated for 187 (56 unique) SNVs and indels. Five multiexon deletions detected by multiple ligation probe assay were assessed from the NGS panel data with ExomeDepth, panelcn.MOPS and CNVPanelizer software.
  
  
  
  There were 48/51 (94%) SNVs and 1/5 (20%) indels consistently detected by all the calling algorithms. Two SNVs were not detected by any of the callers because of a rare reference allele, and one SNV in a low coverage region was only detected by two algorithms. Regarding CNVs, ExomeDepth detected 5/5 multi-exon deletions, panelcn.MOPs 4/5 and only 3/5 deletions were accurately detected by CNVPanelizer.
  
  
  
  The calling efficiency of NGS algorithms for SNVs is influenced by variant type and coverage. NGS protocols need to account for the presence of rare variants in the reference sequence as well as for ambiguities in indel calling. CNV detection algorithms can be used to identify large deletions from NGS panel data for diagnostic applications; however, sensitivity depends on coverage, selection of the reference set and deletion size. We recommend the incorporation of several variant callers in the NGS pipeline to maximize variant detection efficiency.
authors.affiliation: Neurogenetics Group, Instituto de Investigación Sanitaria de Santiago (IDIS), Hospital Clínico de Santiago, level-2, Travesía da Choupana s/n, 15706, Santiago de Compostela, Spain.|Grupo de Medicina Xenómica, CIBERER-U711, Santiago de Compostela, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Cacheiro
authors.affiliation: Neurogenetics Group, Instituto de Investigación Sanitaria de Santiago (IDIS), Hospital Clínico de Santiago, level-2, Travesía da Choupana s/n, 15706, Santiago de Compostela, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Ordóñez-Ugalde
authors.affiliation: Neurogenetics Group, Instituto de Investigación Sanitaria de Santiago (IDIS), Hospital Clínico de Santiago, level-2, Travesía da Choupana s/n, 15706, Santiago de Compostela, Spain.|Grupo de Medicina Xenómica, CIBERER-U711, Santiago de Compostela, Spain.
authors.forename: B
authors.initials: B
authors.lastname: Quintáns
authors.affiliation: Neurogenetics Group, Instituto de Investigación Sanitaria de Santiago (IDIS), Hospital Clínico de Santiago, level-2, Travesía da Choupana s/n, 15706, Santiago de Compostela, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Piñeiro-Hermida
authors.affiliation: Grupo de Medicina Xenómica, CIBERER-U711, Santiago de Compostela, Spain.|Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Amigo
authors.affiliation: Neurogenetics Group, Instituto de Investigación Sanitaria de Santiago (IDIS), Hospital Clínico de Santiago, level-2, Travesía da Choupana s/n, 15706, Santiago de Compostela, Spain.|Grupo de Medicina Xenómica, CIBERER-U711, Santiago de Compostela, Spain.
authors.forename: M
authors.initials: M
authors.lastname: García-Murias
authors.affiliation: Servicio de Neuropediatría, Hospital Universitario La Paz, Madrid, Spain.
authors.forename: S I
authors.initials: SI
authors.lastname: Pascual-Pascual
authors.affiliation: Unidad de Trastornos del Movimiento, Instituto de Investigación Sanitaria Gregorio Marañón, Hospital General Gregorio Marañón, Madrid, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Grandas
authors.affiliation: Servicio de Neurología, Hospital Universitario La Paz, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Arpa
authors.affiliation: Grupo de Medicina Xenómica, CIBERER-U711, Santiago de Compostela, Spain.|Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Carracedo
authors.affiliation: Neurogenetics Group, Instituto de Investigación Sanitaria de Santiago (IDIS), Hospital Clínico de Santiago, level-2, Travesía da Choupana s/n, 15706, Santiago de Compostela, Spain. ssobrido@gmail.com.|Grupo de Medicina Xenómica, CIBERER-U711, Santiago de Compostela, Spain. ssobrido@gmail.com.
authors.forename: M J
authors.initials: MJ
authors.lastname: Sobrido
mesh_terms: D056915:DNA Copy Number Variations; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D035583:Rare Diseases / Q000235:genetics; D015419:Spastic Paraplegia, Hereditary / Q000235:genetics*