Primary data were filtered accordingly to signal purity with the Illumina Real-Time Analysis (RTA) software v1.8. Reads were mapped against the human reference genome built 19 (hg19) using the Burrows-Wheeler-Aligner tool (bwa). Paired end reads were fixed by PICARD. Several steps to enhance data-quality were carried out using GATK v3.3, e.g. local realignment around short insertion and deletions and recalibration of the base quality scores. The GATK HaplotypeCaller was used to identify SNPs and INDELs. Subsequently, variant re-calibration was performed using stochastic models and divers training-sets (hapmap_3.3.hg19, 1000G_omni2.5.hg19, dbsnp_138.hg19,1000G_phase1.snps.high_confidence.hg19,Mills_and_1000G_gold_standard.indels.hg19). Detected variants were then annotated with the help of ANNOVAR. Specific filter-criteria were applied: focus on exonic and splicing regions; allele frequency of less than 0.01 in dbSNP, the 1000-Genomes project, the Exome Variant Server or the ExAC browser and in an in-house database; and prioritization for de novo mutations and compound-heterozygous / homozygous variants.
Real time analysis software v1
Manufactured by Illumina
The Real-Time Analysis (RTA) software v1.8 is a core component of the Illumina sequencing platform. Its primary function is to process and analyze data generated during the sequencing run in real-time. The software is responsible for performing base calling, image analysis, and other essential steps required to generate high-quality sequencing data.
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Lab products found in correlation
2 protocols using real time analysis software v1
1
Variant Identification and Annotation Pipeline
2
Exome Sequencing and Genetic Variant Identification
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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.
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