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Protocol publication

[…] Agilent SureSelect Human All Exon 50 Mb versions 3, 4, and 5 (Agilent Technologies Santa Clara, CA) were used for in-solution enrichment of coding exons and flanking intronic sequences following the manufacturer's standard protocol. The enriched DNA samples were subjected to standard sample preparation for the HiSeq 2000 instrument (Illumina San Diego, CA). The Illumina CASAVA v1.8 pipeline was used to produce 99 bp sequence reads. BWA was used to align sequence reads to the human reference genome (hg19) and variants were called using the GATK ( software package. All single nucleotide variants (SNVs) and insertion/deletions (INDELs) were submitted to SeattleSeq137 for further characterization and annotation. Sanger sequencing was used for confirmation and segregation of the variants in each family. [...] We analyzed WES data using our in house tool ( Our workflow is seen in . The analysis started with QC checks including the coverage and average read depth of targeted regions, numbers of variants in different categories, and quality scores. All variants were annotated and categorized into known and novel variants. As previously recommended, we filtered variants based on minor allele frequency of <0.005 in dbSNP141. We also filtered out variants that are present in >10 samples in our internal database of >3,000 exomes from European, Asian, and American ancestries that includes Turkish, Iranian, Mexican, Ecuadorian, and Puerto Rican samples (). Autosomal recessive inheritance with both homozygous and compound heterozygous inheritance models, and a genotype quality (GQ) score >35 for the variant quality were chosen. Missense, nonsense, splice site, in-frame INDEL and frame-shift INDELs in the known ARNSD genes () were selected. Missense variants that remained after these filters were later analyzed for presence in the Human Gene Mutation Database (HGMD) ( and having a pathogenic prediction score at least in two of the following tools: PolyPhen2, SIFT, MutationAssessor, and MutationTaster. Finally, we used CoNIFER (Copy Number Inference From Exome Reads) and XHMM (eXome-Hidden Markov Model) to detect CNVs. After this filtering, only those variants co-segregated with the phenotype in the entire family was considered pathogenic. […]

Pipeline specifications

Software tools BaseSpace, BWA, GATK, PolyPhen, SIFT, Mutationassessor, MutationTaster, CoNIFER, XHMM
Databases dbSNP HGMD
Organisms Meleagris gallopavo
Diseases Deafness, Wiskott-Aldrich Syndrome, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Hernias, Diaphragmatic, Congenital