Targeted NGS for Variant Discovery

Genomic DNA was extracted from peripheral blood samples using the blood genomic extraction kit (Qiagen, Germany) and subjected to targeted next-generation sequencing. The coding exons and flanking sequences of over 20,000 genes were captured for paired-end sequencing (2× 100 bp) on Illumina HiSeq2500 (Illumina, USA). The average sequencing depth of the target region was higher than 170×, with over 95% of bases covered by at least 30×. An in-house genome analysis pipeline composed of BWA 0.7.12 (Li and Durbin, 2009 (link)), GATK 3.5 (McKenna et al., 2010 (link); DePristo et al., 2011 (link)), snpEff 4.0 (Cingolani et al., 2012 (link)), BED Tools 2.25.0 (Quinlan, 2014 ), ANNOVA (Yang and Wang, 2015 (link)) were used to process demultiplexed fastq data. BAM files were generated to visualize read pairs and variant calling in IGV (Broad Institute, USA). Candidate variants were filtered based on the population frequency of 2%, predicated variant pathogenicity, inheritance mode, and protein interaction network.

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Chen Y., Yang B., Zhang X.M., Chen S., Wang M., Hu L., Pan N., Li S., Shi W., Yang Z., Wang L., Tan Y., Wang J., Wang Y., Xing Q., Ma Z., Li J., Huang H.F., Zhang J, & Xu C. (2023). Biallelic variants in RBM42 cause a multisystem disorder with neurological, facial, cardiac, and musculoskeletal involvement. Protein & Cell, 15(1), 52-68.

Publication 2023

blood genomic extraction kit HiSeq2500

Corresponding Organization :

Other organizations : Fudan University, International Peace Maternity & Child Health Hospital, Shanghai Jiao Tong University, Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Obstetrics and Gynecology Hospital of Fudan University, China National Rice Research Institute, Zhejiang University, Baylor College of Medicine, Children's Hospital of Fudan University, ShanghaiTech University, Chinese Academy of Medical Sciences & Peking Union Medical College

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Variable analysis

independent variables

Genomic DNA extraction method (blood genomic extraction kit, Qiagen, Germany)
Targeted next-generation sequencing (coding exons and flanking sequences of over 20,000 genes were captured for paired-end sequencing (2× 100 bp) on Illumina HiSeq2500)

dependent variables

Sequencing depth of the target region (average higher than 170×, with over 95% of bases covered by at least 30×)
Candidate variants (filtered based on population frequency, predicted variant pathogenicity, inheritance mode, and protein interaction network)

control variables

Peripheral blood samples

controls

No positive or negative controls were explicitly mentioned in the provided information.

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