Ez human exome library v2.0 kit

Exome sequencing was performed in individual VI:3 in family 1. Briefly, genomic DNA was enriched for exonic and adjacent splice site sequences with the SeqCap EZ human exome library v2.0 kit, and libraries were run on an Illumina HiSeq 2000 sequencer via a paired-end 100-bp protocol (Hussain et al. 2013 (link)). For data analysis, the Cologne Center for Genomics (CCG) Varbank pipeline v2.6 and user interface was used (Kawalia et al. 2015 (link)). Primary data were filtered according to signal purity by the Illumina realtime analysis (RTA) software v1.8. Subsequently, the reads were mapped to the human genome reference build GRCh37/hg19 (http://www.genome.ucsc.edu/) using the BWA-SW alignment algorithm. Mean coverage was 100× in the exome, and 95.6% and 86.4% of target bases were covered more than 10× and 30×, respectively. Further annotation and filtering for high-quality rare variants (MAF < 0.1%) with a predicted impact on protein sequence or splicing was performed as previously described (Borck et al. 2015 (link)). We also filtered against an inhouse database containing variants from 511 exomes from individuals with epilepsy to exclude pipeline-related artifacts.

A 100 ng/μl gDNA of two members (IV-1 and IV-4) was used for exome sequencing. After preparing the sequencing libraries using the SeqCap EZ human exome library v2.0 kit, the Illumina HiSeq 4000 sequencing machine via a paired-end 100-bp protocol (Hussain et al., 2013) was used for sequencing. Illumina real-time analysis (RTA) software v1.8 was used to filter the primary data, followed by the human reference genome build GRCh37/hg19 (http://www.genome.ucsc.edu/) for mapping the reads, using the BWA-SW alignment algorithm. Picard tools and the Genome Analysis Toolkit (GATK) were used to improve the reads quality for realignment and base quality score recalibration. The Platypus, Haplotype Caller, and Mpileup programs were used to perform the calling of single nucleotide polymorphisms (SNPs) and short insertions/deletions (INDELs). The variant quality score calibration (VQSR) using GATK was used for further filtration. The ALLEGRO program identified the large runs of homozygosity (ROH) based on multipoint linkage analysis. The CNMOPS and ExomeDepth algorithms were utilized to check the coverage of CNVs, and the variants data was combined and annotated using the COMBINE and FUNC algorithms.