Whole Exome Sequencing
Genomic DNA was extracted from peripheral blood samples according to
standard methods. WES was performed using the Whole Exome Solution kit
by Sophia Genetics, SA, following the manufacturer’s recommendations.
The resulting libraries were subjected to paired-end sequencing on an
Illumina NextSeq 500 platform. Library preparation and sequencing were
outsourced (Genotypos-Science Labs, Athens). Raw data were converted to
FastQ files for computational analysis which included read alignment to
the human genome reference sequence (GRCh37/hg19), duplicate marking,
base quality score recalibration, indel realignment and variant calling,
using the SOPHiA DDM® platform (Sophia Genetics, SA). Variant
annotation with ANNOVAR algorithm (K. Wang et al., 2010) and variant
filtration process were performed using VarAFT v2.16 (http://varaft.eu)
application (Desvignes et al., 2018).
Variant filtering involved a six-step phenotypic-driven strategy as
presented in Figure 2 and summarized as follows: (1) Variant filtering
by phenotype and/or in silico gene list; (2) Variant filtering by
population frequency (Minor Allele Frequency, MAF ≤1%); (3) Variant
filtering by variant type and/or ClinVar pathogenicity and/or in-silico
pathogenic prediction tools; (4) Variant filtering by genotype; (5)
Variant filtering by American College of Medical Genetics and Genomics
(ACMG) classification; (6) Clinical/phenotypic (re)evaluation of
retained variant(s).
The accepted WES data quality control parameters included a mean depth
of coverage >50x, with >98% regions at 25x.
Compared to the human genome reference hg19, ~30.000
variants were identified per exome including only exonic and flanking
(+/- 10bp) regions. Subsequent to variant filtration using criteria
outlined in Fig. 1, distinct variants were selected and set for further
phenotype/genotype assessment and evaluation by a multidisciplinary team
including the referring clinician