Sureselect clinical research exome v2

Genomic DNA extracted from fresh blood cells from the proband and her father with QiAmp DNA mini kit (Qiagen) was captured using Agilent in-solution enrichment methodology with their biotinylated oligonucleotides probes library (SureSelect Clinical Research Exome V2, Agilent Technologies), followed by paired-end 75 bases massively parallel sequencing on Illumina HiSeq4000 (IntegraGen SA, Evry, France), as reported (13 (link)). Sequence capture, enrichment and elution were performed according to manufacturer’s instruction and protocols (SureSelect, Agilent) without modification, except for library preparation performed with NEBNext® Ultra kit (New England Biolabs). For library preparation, 600 ng of each genomic DNA were fragmented by sonication and purified to yield fragments of 150–200 bp. Paired-end adaptor oligonucleotides were ligated on repaired fragments then purified and enriched by 8 PCR cycles. A total of 1200ng of the purified Libraries were then hybridized to the SureSelect oligo probe capture library for 72 h. After hybridization and washing, the eluted fraction was PCR-amplified, purified and quantified by qPCR. Each eluted-enriched DNA library was then sequenced on an Illumina HiSeq4000 as paired-end 75 bp reads. Image analysis and base calling were performed using Illumina Real Time Analysis (2.7.7) with default parameters.

A bionix (60 (link)) pipeline was used to process samples from sequencing data to variant calls. Sequences were aligned to Genome Reference Consortium Human Build 38 (GRCh38) using minimap2 v2.17 (61 (link)), and also to Mus Musculus reference GRCm38. Mouse derived sequences were removed with XenoMapper v1.0.2 (62 (link)). PDX whole exome sequencing used the Agilent SureSelect Clinical Research Exome V2, with reads filtered to 100bp each side of capture regions. Small mutations were called using Octopus v0.7.0 (63 (link)) and annotated using SnpEff v4.3 (64 ). Copy Number Variation was estimated using FACETS v0.6.1 (65 (link)).

Variant calling for exome-sequencing alignments was performed with Mutect 2⁷⁴ (GATK v. 4.1.3.0) using the parameters “–genotype-germline-sites true–genotype-pon-sites true–af-of-alleles-not-in-resource 0.0000025–disable-read-filter MateOnSameContigOrNoMappedMateReadFilter”. The GnomAD^{75 (link)} population variant database was provided as a germline resource, together with the same reference genome as above. The analysis was restricted to exome target regions corresponding to Agilent SureSelect Clinical Research Exome v2 or Twist Exome, depending on sequencing batch. In addition, a panel of normals was supplied as input, built from all available normal samples in the study. This panel was built by first running Mutect 2 in tumor-only mode on each normal with the parameter “–disable-read-filter MateOnSameContigOrNoMappedMateReadFilter” and then running CreateSomaticPanelOfNormals (GATK) on the resulting files. Variant-quality labels were assigned using FilterMutectCalls (GATK) using the same reference genome as previously. These variants were then annotated using the script vcf2maf.pl (https://github.com/mskcc/vcf2maf), which relies on VEP^{76 (link)}, using the v. 98 build of the VEP reference database for the GRCh37 genome.

Variant calling for exome sequencing alignments was performed with Mutect 2 [6 (link)] (GATK v. 4.1.3.0) using the parameters “—genotype-germline-sites true —genotype-pon-sites true —af-of-alleles-not-in-resource 0.0000025 —disable-read-filter MateOnSameContigOrNoMappedMateReadFilter”. The GnomAD [7 (link)] population variant database was provided as a germline resource, together with the same reference genome as above. The analysis was restricted to exome target regions corresponding to Agilent SureSelect Clinical Research Exome v2. Variant qualities were further assessed using FilterMutectCalls (GATK). These variants were then annotated using the script vcf2maf.pl (https://github.com/mskcc/vcf2maf, accessed on 2 May 2019), which relies on VEP, using the v. 98 build of the VEP reference database for the GRCh37 genome. Variants were further filtered using custom scripts to remove genes with >0.001 frequency in GnomAD, ExAC, and genes with dbSNP identifiers, unless any of these variants were whitelisted. Variants were whitelisted if they were either listed as oncogenes in Cancer Gene Census (CGC) and the exact mutation was listed in COSMIC or if they were listed as tumour suppressors in CGC. The resulting list was further filtered to remove variants that only occurred in more than one PDX sample but not in any patient biopsy.

The samples of the affected individuals were subjected to ES of parent–child trios after the informed consent was obtained. Some of their unaffected family members as potential living donors were selected for ES post informed consent. ES procedure and variants annotation have been described in detail previously^{12 (link)}. Genomic DNA was isolated from blood lymphocytes and was fragmented to an average size of 250 bp. End repair, adapter ligation, and PCR enrichment were performed following the protocol for VAHTS TM Universal DNA Library Prep Kit for Illumina V3 (Vazyme Biotech Co., Ltd, Nanjing, China). The enriched DNA libraries were subjected to exome capture using Agilent SureSelect Clinical Research Exome V2 or Human All Exon V7. The resulting libraries were sequenced on Illumina sequencers (HiSeq 4000 or Hiseq X) with the paired-end of 150 bp at Precision Medicine Center of Zhengzhou University. Variant interpretation was performed manually by a panel of nephrologists and clinical molecular geneticists. For clinical sequence interpretation the variants were classified according to the ACMG guidelines^{16 (link)}.

Peripheral blood samples were collected from the proband and his family. A total of 1.0 μg of genomic DNA from each sample was sheared into 200–300 bp fragments, which were subsequently analyzed with a SureSelect clinical research exome V2 (Agilent, USA) exon capture kit following the official guide. Sequencing was performed on the Illumina NovaSeq 6000 platform with a 150PE sequencing strategy.
After removing low‐quality reads and sequencing adapters, the clean data were mapped to the human reference genome (hg19) by the Burrows–Wheeler Aligner (Li & Durbin, 2009 (link)). SNPs and short insertions or deletions were analyzed by GATK. Variants were annotated with ANNOVAR (Wang et al., 2010 (link)), and those located in exonic and splicing regions with a minor allele frequency (MAF) ≤0.0001/0.005 in the SNP database (Exome Aggregation Consortium [ExAC], 1000 Genomes, gnomAD) were obtained for further analysis. The pathogenicity of the variants was evaluated according to the ACMG guidelines (Richards et al., 2015 (link)). Candidate pathogenic variants were further validated by Sanger sequencing. All the variants detected were filtered by MAF, and the variant types (exonic and splicing) are listed in Table S1.