Agilent sureselect human all exon v6 kit

Genomic DNA was extracted from brain tissues by Blood & Tissue Genomic DNA Extraction Kit (Tiangen). Sequencing libraries were generated using Agilent SureSelect Human All Exon kit V6 (Agilent Technologies, CA, USA) following the manufacturer’s recommendations. Briefly, fragmentation was carried out by the hydrodynamic shearing system (Covaris, Massachusetts, USA) to generate short fragments. The sequencing libraries were constructed on a cBot Cluster Generation System using Hiseq PE Cluster Kit (Illumina) according to the manufacturer’s instructions. Libraries were subjected to 150 bp paired-end sequenced on the Illumina NovaSeq 6000. Each sample was sequenced to 400× coverage with an average of 161 million (M) reads (SD = 21 M).

Genomic DNA of samples was extracted by QIAamp DNA Mini Kit (Qiagen) and then captured with an Agilent SureSelect Human All Exon Kit V6 (Agilent). DNA libraries were generated following the protocols provided by Illumina. DNA libraries were sequenced with the Illumina NovaSeq 6000 System (Illumina), yielding 150 bp of paired-end sequence, and FASTQ files were generated. The WES sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

Library preparation, hybridization capture, and WES procedures were performed by Novogene Corporation Inc. (Sacramento, California, USA). Sequencing libraries from the extracted DNA were generated using the Agilent SureSelect Human All Exon Kit V6 (Agilent, Santa Clara, California, USA, 5190-8865) following the manufacturer’s recommendations. Products were purified using the AMPure XP system (Beckman Coulter, Brea, California, USA, A63882) and quantified using an Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system. The post-capture libraries were sequenced on the Illumina NovaSeq 6000 platform.

Genomic DNA of the entire subjects was extracted from whole blood, using the Qiagen Blood DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s directive. The genomic DNA of four affected patients and unaffected family members from the RP family were subjected to WES analysis. The whole-exome was captured by using Agilent SureSelect Human All Exon Kit V6 (Agilent Technologies, Santa Clara, CA, USA). The HiSeq 2500 (Illumina, San Diego, CA, USA) platform was used for paired-end sequencing with reading lengths of 150 bp and used a minimum of 20 million reads per sample.

Blood samples were collected from the peripheral blood of individuals into tubes containing EDTA. DNA extraction was carried out using the RelaxGene Blood DNA System Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. For the GWAS, all samples were genotyped individually using Illumina Infinium OmniZhongHua-8v1-3_A1 by the BioMiao Biological Technology Company (Beijing, China). For the WES, 3 μg of purified gDNA was fragmented to 180–280 bp and subjected to DNA library creation using established Illumina paired-end protocols. The Agilent SureSelect Human All ExonV6 Kit (Agilent Technologies, Santa Clara, CA, United States) was used for exome capture according to the manufacturer’s instructions. The Illumina Novaseq 6000 platform (Illumina Inc., San Diego, CA, United States) was utilized for genomic DNA sequencing by Novogene Bioinformatics Technology Co., Ltd (Beijing, China) to generate 150-bp paired-end reads with a minimum coverage of 10× for ∼99% of the genome (mean coverage of 100×).

DNA was extracted from 70 GC tumors and paired normal gastric mucosa. QIAamp DNA Mini Kit (Qiagen) was used to isolate genomic DNA from tumor tissues and matched normal mucosa according to the manufacturer’s instructions. Then we combined the following two steps to verify the quality of isolated genomic DNA. First, DNA degradation and contamination were monitored on 1% agarose gels. Second, Qubit® DNA Assay Kit in Qubit® 2.0 Flurometer (Invitrogen, USA) was used to quantify DNA concentration.
Whole-exome library construction was generated using the Agilent SureSelect Human All Exon V6 Kit (Agilent Technologies, Santa Clara, CA, USA). The index-coded samples were clustered on a cBot Cluster Generation System using Hiseq PE Cluster Kit (Illumina). Then the DNA libraries were sequenced on Illumina Hiseq platform (Illumina, San Diego, California, USA) and 150 bp paired-end reads were generated. We first conducted data quality control and then performed all downstream bioinformatics analyses based on the high-quality clean data, in which reads containing an adapter, reads containing poly-N, and low-quality reads were removed. The paired-end clean reads were aligned to the Human Genome Reference Consortium build 37 (GRCh37) using BWA v.0.7.8 [22 (link)]. Mapped reads were then de-duplicated using Sambamba tools (v0.4.7) [23 (link)].