Computational protocol: A patient with van Maldergem syndrome with endocrine abnormalities, hypogonadotropic hypogonadism, and breast aplasia/hypoplasia

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Protocol publication

[…] Our molecular genetic studies were prospectively reviewed and approved by the Institutional Review Board (IRB) of The Research Institute at Nationwide Children’s Hospital. Consent was obtained from the patient and parents for whole exome sequencing (WES). All genomic DNA samples in the study were processed using Agilent SureSelectQXT Target Enrichment System for Illumina Paired End Sequencing Protocol (Agilent Technologies, CA). Genomic DNA was sheared and target exonic regions were captured with the Agilent SureSelect Clinical Research Exome kit. Paired-end 151 base pair reads were generated for exome-enriched libraries sequenced on the Illumina HiSeq 4000 (Illumina, CA). All samples were sequenced to a depth of 100X.Secondary analysis was performed using Churchill, a pipeline developed in house for the discovery of human genetic variation []. Churchill utilizes the Burrows-Wheeler Aligner (BWA) to align sequence data to the reference genome (build GRCh37). Further refinement steps were performed on the aligned sequence data following the Broad Institutes guidelines for best practices (https://www.broadinstitute.org/gatk/guide/best-practices). Duplicate sequence reads were removed using PicardTools (v1.117). Local realignment was performed on the aligned sequence data using the Genome Analysis Toolkit (v3.4–0). Churchill’s own deterministic implementation of base quality score recalibration was used. The GATK’s HaplotypeCaller (HC) was used to call variants. To maximize sensitivity, variant calling was performed across all samples in the study. Use of the GATK’s variant quality score recalibration (VQSR) was excluded in favor of using Churchill’s own quality-based variant filtering algorithm. Comparison of variant calling performance with the NIST Genome in a Bottle (GIAB) gold reference standard (version 3.22) utilizing whole genome sequencing data for NA12878 (30X) demonstrated that Churchill is 99.9985% accurate, with a sensitivity (recall) of 99.6061%.Tertiary analysis was performed using ANNOVAR and custom in-house scripts to annotate the variant call set with mutation and gene information, protein functional predictions, and population allele frequencies []. Common variation occurring at greater than 1% mean allele frequency (MAF) in the population was excluded, and variants outside of coding regions (defined as greater than 4 base pairs from an exon splice site) as well as exonic variants coding for synonymous single nucleotide polymorphisms were also dropped. Variants were further filtered based on the pattern of inheritance expected from examination of the pedigree. […]

Pipeline specifications

Software tools BWA, GATK, Picard, ANNOVAR
Applications WGS analysis, WES analysis