Genomic DNA was extracted from peripheral blood leukocytes according to standard procedures, and then exome libraries (Agilent SureSelectXT Reagent Kit; Agilent Technologies) were sequenced on an Illumina HiSeq 2500 at the Genomic Technologies Facility in Lausanne, Switzerland. Bioinformatic analyses were performed as described before.5 (link) Briefly, raw reads were mapped to the human reference genome (hg19/GRCh37) using the Novoalign software (V3.08.00, Novocraft Technologies). Next, Picard (version 2.14.0-SNAPSHOT) was used to remove duplicate reads and GATK was used (version 3.8)6 (link) to perform ‘base quality score recalibration’ on both single nucleotide variants and insertion-deletions. A VCF file with the variants was generated by HaplotypeCaller. Then, DNA variants were filtered based on quality, frequency in ExAC, gnomAD,7 (link) 1000 Genomes,8 (link) ESP (NHLBI Exome Variant Server, http://evs.gs.washington.edu/EVS ), GME (GME Variome http://igm.ucsd.edu/gme/index.php ) and ABraOM,9 (link) and on predicted impact on protein sequence and mRNA splicing. Finally, they were annotated according to a specific in-house pipeline.5 (link)
Protocol detail
Exome Sequencing of Peripheral Blood Samples
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Exome
Genome human
Genomes
Human
Insertion deletions
Nucleotide
Peripheral blood leukocytes
Protein sequence
Corresponding Organization : University of Basel
Other organizations : Centro de Genética Clínica, Hospitais da Universidade de Coimbra, University of Coimbra, Boston Children's Hospital, Harvard University, Instituto de Medicina Integral Professor Fernando Figueira, Universidade de Pernambuco, Yokohama City University, Howard Hughes Medical Institute, Stanford University
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Variable analysis
independent variables
- None explicitly mentioned
- Genomic DNA extracted from peripheral blood leukocytes
- Exome sequencing using Illumina HiSeq 2500 platform
- Bioinformatic analysis of sequencing data, including read mapping, duplicate removal, base quality score recalibration, variant calling, and variant filtering
- Standard procedures for genomic DNA extraction from peripheral blood leukocytes
- Use of Agilent SureSelectXT Reagent Kit for exome library preparation
- Sequencing performed at the Genomic Technologies Facility in Lausanne, Switzerland
- Bioinformatic analysis pipeline described in a previous publication (reference 5)
- No positive or negative controls were explicitly mentioned in the input protocol
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