Comparison of two next-generation sequencing kits for diagnosis of epileptic disorders with a user-friendly tool for displaying gene coverage, DeCovA

Abstract

In recent years, molecular genetics has been playing an increasing role in the diagnostic process of monogenic epilepsies. Knowing the genetic basis of one patient's epilepsy provides accurate genetic counseling and may guide therapeutic options. Genetic diagnosis of epilepsy syndromes has long been based on Sanger sequencing and search for large rearrangements using MLPA or DNA arrays (array-CGH or SNP-array). Recently, next-generation sequencing (NGS) was demonstrated to be a powerful approach to overcome the wide clinical and genetic heterogeneity of epileptic disorders. Coverage is critical for assessing the quality and accuracy of results from NGS. However, it is often a difficult parameter to display in practice. The aim of the study was to compare two library-building methods (Haloplex, Agilent and SeqCap EZ, Roche) for a targeted panel of 41 genes causing monogenic epileptic disorders. We included 24 patients, 20 of whom had known disease-causing mutations. For each patient both libraries were built in parallel and sequenced on an Ion Torrent Personal Genome Machine (PGM). To compare coverage and depth, we developed a simple homemade tool, named DeCovA (Depth and Coverage Analysis). DeCovA displays the sequencing depth of each base and the coverage of target genes for each genomic position. The fraction of each gene covered at different thresholds could be easily estimated. None of the two methods used, namely NextGene and Ion Reporter, were able to identify all the known mutations/CNVs displayed by the 20 patients. Variant detection rate was globally similar for the two techniques and DeCovA showed that failure to detect a mutation was mainly related to insufficient coverage.

Exome sequencing identifies recurrent BCOR alterations and the absence of KLF2 , TNFAIP3 and MYD88 mutations in splenic diffuse red pulp small B-cell lymphoma

“…(A) Copy number (CN) variation was detected from exon sequencing of the target genes after normalization using DeCovA. 25 , 24 Each row represents a lymphoma case (HCL: hairy-cell lymphoma; SDRPL: splenic diffuse red pulp lymphoma; SMZL: splenic marginal zone lymphoma). Each column represents an exon of two different genes ( BCOR and KDM6A ) located at locus Xp11.…”

“…The procedure for detecting copy number variation was adapted from previously published methods; read depths of each exon from all of the target genes were obtained for each sample using DeCovA. 24 , 25 Exon read depths (ERD) were then normalized against the sum of all ERD from the same sample. A copy number ratio (CNR) was obtained by dividing the ERD of the sample by the median ERD of all of the samples as a control: CNR x =(ERD x /∑ERD 1>n ) sample /(ERD x /∑ERD 1>n ) control .…”

Whole MYBPC3 NGS sequencing as a molecular strategy to improve the efficiency of molecular diagnosis of patients with hypertrophic cardiomyopathy

“…VCF metrics were collected using snpEff/SnpSift (Cingolani et al, ). DeCovA, an in‐house script, was used for copy number variant detection (Dimassi et al, ).…”

Primary ciliary dyskinesia gene contribution in Tunisia: Identification of a major Mediterranean allele

“…Common polymorphisms (allele frequency ≥ 0.8% in the general population) were filtered‐out as benign variants based on 1000 Genomes (http://www.internationalgenome.org/1000-genomes-browsers), Exome Variant Server (http://evs.gs.washington.edu/EVS/), and gnomAD (http://gnomad.broadinstitute.org/) databases. Large deletions/duplications (spanning more than one exon) were searched by read depth ratio analysis of NGS data (Dimassi et al, ). Homozygosity was confirmed by the above‐mentioned read depth ratio analysis that highlighted the existence of two copies for the relevant exon.…”