Computational protocol: Diagnosis of lethal or prenatal‐onset autosomal recessive disorders by parental exome sequencing

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Protocol publication

[…] Genomic DNA samples were quantified according to the manufacturer's instructions on the Qubit fluorimeter (Thermo Fisher Scientific, Massachusetts, USA) to determine that the minimum quantity of DNA required, 3000 ng, was available. The samples were fragmented by using the Bioruptor (Diagenode, Liège, Belgium) and indexed adaptors ligated before hybridization with the Agilent SureSelect All Exon capture kit (v4, v5, or v6) or Agilent SureSelect Focused exome kit (Santa Clara, CA, USA). Paired‐end 100‐bp reads were sequenced on a HiSeq 2500 (Illumina, San Diego, CA, USA) by using either the standard or the rapid run mode or paired‐end 150‐bp reads on the NextSeq500 (Illumina, San Diego, CA, USA) by using either a mid or high output flow cell. Approximately 12 whole exomes can be run per flow cell, to generate at least 60 million reads with >80X mean coverage and >98% of target bases at ≥20X. Samples were sequenced in multiple batches as and when samples were received for diagnostic testing. The Illumina HiSeq FASTQ sequencing reads were demultiplexed and aligned to the reference (GRCh37/Hg19) by using BWA‐MEM (v0.7.12), converted to BAM format file and subjected to duplicate removal by using Picard (v1.129). GATK (v3.4‐46) was used for indel realignment, variant calling, and quality filtering. [...] Variants were annotated by using Alamut‐Batch (v1.4.4), a variant call format file was inputted and all SNVs and indels were annotated by using a range of different variant and genomic databases, including HGMD Professional. A bioinformatics pipeline was designed in‐house to identify shared genes where both parents had a heterozygous potentially pathogenic variant and to identify X‐linked recessive variants where appropriate (Figure ). Variants with a MAF < 0.0001 (<0.01%) and 0.001 (<0.1%) in Exome aggregation consortium (ExAC or the Exome variant server (EVS were retained to produce a subset of very rare variants and rare variants. Variants were restricted to nonsynonymous variants, those affecting the conserved splice sites or those within −50/+10 base pairs of flanking exons predicted by Alamut‐Batch to affect splicing (5 tools were used: SpliceSiteFinder‐like, MaxEntScan, NNSplice [Fruitfly], GeneSplicer, and Human Splicing Finder). Variants annotated as Pathogenic in HGMD Pro (all cases) or ClinVar (since 2016) were retained regardless of other filtering criteria. Copy number variants were identified by using read depth analysis with a modified version of R software package ExomeDepth (v1.1.8) and comparing the test sample against reference samples. Autosomal recessive variants were identified where parents either shared the same heterozygous variant or had different heterozygous variants in the same gene. Potential X‐linked recessive variants in the mother were also shortlisted where only male pregnancies were affected. The bioinformatics pipeline is summarized in Figure . [...] All genes on the variant shortlist were considered and initially reviewed via the Online Mendelian Inheritance in Man database (OMIM and PubMed ( Candidate variants in known disease‐causing genes were identified for further investigation by comparison with the fetal phenotype. Previous reports of a variant were determined by using HGMD professional, ClinVar ( and Locus‐specific databases. In silico tools were accessed via Alamut Visual (versions 2.7.2‐2.10) to predict pathogenicity of variants. Likely causative variants identified in this series were variants that were reported as likely pathogenic or pathogenic in the patient's clinical diagnostic report. We have reviewed all the variant classifications by using the recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology guidelines. In cases where no likely diagnosis was identified, we undertook further analysis of a curated gene panel from PanelApp ( […]

Pipeline specifications

Software tools BWA, Picard, GATK, Alamut Batch, MaxEntScan, NNSplice, GeneSplicer, ExomeDepth, Alamut Visual, PanelApp
Databases ClinVar LSDBs Exome Variant Server OMIM HGMD
Applications Genome annotation, WGS analysis, WES analysis
Organisms Homo sapiens, Mus musculus
Diseases Genetic Diseases, Inborn