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.
Realtime analysis rta software v1
Manufactured by Illumina
Illumina's RTA software v1.8 is a core component of the sequencing analysis pipeline. It performs real-time analysis of data generated during the sequencing run, including image analysis, base calling, and quality scoring. The RTA software is designed to provide fast and accurate processing of sequencing data to enable efficient and reliable downstream analysis.
Automatically generated - may contain errors
2 protocols using realtime analysis rta software v1
1
Exome Sequencing for Rare Genetic Variants
2
Exome Sequencing Analysis for Genetic Variants
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A 100 ng/μl DNA of two members (IV-1 and IV-4) was used for exome sequencing. The sequencing libraries of the DNA were prepared with the SeqCap EZ human exome library v2.0 kit. The sequencing was done on Illumina HiSeq 4000 sequencing machine via a paired-end 100-bp protocol (Hussain et al., 2013 (link)). The filtration of primary data was carried out by the Illumina real-time analysis (RTA) software v1.8. Afterward, the mapping of the reads to the human reference genome build GRCh37/hg19 (http://www.genome.ucsc.edu/ ) was performed using the BWA-SW alignment algorithm. Picard tools were used to improve the read quality, and Genome Analysis Toolkit (GATK) was used for realignment and base quality score recalibration. The calling of single nucleotide polymorphisms (SNPs) and short insertions/deletions (INDELs) was performed by Platypus, Haplotype Caller, and Mpileup programs and further filtration was carried out through variant quality score calibration (VQSR) using GATK. The ALLEGRO program identified several large runs of homozygosity (ROH) based on multipoint linkage analysis. For the coverage analysis of CNV detection, CNMOPS and ExomeDepth algorithms were utilized. In addition, the COMBINE and FUNC algorithms were used to combine the data and the annotation of functional variants.
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