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Pipeline publication

[…] herlands). Fluorescence in situ hybridization (FISH) was also use in order to determine whether the rearrangement had been inherited as a balanced translocation. The FISH was performed according to standard protocols., We extracted DNA from whole blood and quantified it with a fluorometer (Qubit; Life Technologies Corporation, Carlsbad, CA, USA). The exome was captured and enriched from genomic DNA using an exome enrichment kit (Nextera; Illumina Inc., San Diego, CA, USA), representing 62 Mbp of the human genome (hg19 build). Sequencing was performed with a high-resolution optical imaging system (HiScan; Illumina Inc.)., Fragments were aligned to the hg19 reference genome through the use of Burrows-Wheeler alignment [], and variant calling was performed with a software package for the analysis of sequence data (Genome Analysis Toolkit; Broad Institute, Cambridge, MA, USA) [–]. A total of 800 informative single nucleotide polymorphisms were analyzed with the whole-genome association analysis toolset (PLINK; to confirm the familial relationship between samples []. For genotype calls (in all members of the quartet, we used only reads with a Phred score ≥ 30 [], bases with ≥ 20 reads, and a genotype quality score ≥ 20 []., Variant annotation was performed with an annotation program (ANNOVAR, []. To select rare variants (minor allele frequency ≤ 0.01) and novel variants, we employed the following databases: 1000 Genomes Project (; the National Heart, Lung, and Blood Institute GO Exome Sequencing Project (6500 exomes;; and the Exome Aggregation Consortium ( Variants with a depth of coverage of less than 20× reads in all members of the family were excluded in order to avoid false positives. To identify de novo variations, we accepted candidates only at loci that were homozygous for the reference allele in the parents. For the gene analysis, we selected only nonsynonymous SNVs, […]

Pipeline specifications

Software tools BWA, GATK, PLINK, ANNOVAR