title: exome-seq of a ccRCC tumor (advanced stage)
design: 500 ng DNA were used for library construction. Sequencing libraries were generated using Agilent SureSelect Human All Exon kit (Agilent Technologies) following manufacturers recommendations and index codes were added to attribute sequences to each sample. DNA samples were sonicated by hydrodynamic shearing system (Covaris) to generate 180-280 bp fragments. Remaining DNA overhangs were converted into blunt ends via exonuclease/polymerase activities, and the enzymes were then removed. After adenylation of 3 ends of DNA fragments, adapter oligonucleotides were ligated. DNA fragments with ligated adapter molecules on both ends were selectively enriched using PCR. After PCR, the library was hybridized in the liquid phase with biotin-labeled probes, after which streptavidin-coated magnetic beads were used to capture the exons of genes. Captured libraries were enriched by PCR to add index tags to prepare for hybridization. Resulting products were then purified using the AMPure XP system (Beckman Coulter) and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system. The qualified libraries were sequenced using Illumina NovaSeq6000 after pooling according to its effective concentration and expected data volume. Filtering raw sequencing reads was performed using Trim Galore software (v0.6.7; 10.5281/zenodo.5127898), parameters: -q 25 phred33 length 36 -e 0.1 stringency 3 fastqqc. Alignment to hg38 was performed with Bowtie2 software (v2.2.5; (1)), parameters: default. After sorting with samtools (v0.1.19) and marking duplicates with Picard in GATK, the read alignment results were finally stored in BAM format.