title: DNA-Seq of blood:girl
design: Genomic DNA of 1 - 3 g was fragmented to an average size of 150 bp using a S220 Focused-ultrasonicator (Covaris, Massachusetts, USA). A DNA Sample Prep Reagent Set (MyGenostics, Beijing, China) was used for the preparation of standard Illumina libraries, including end repair, adapter ligation, and PCR amplification, which would be further sequenced by illumina (NOVESEQ6000). Enrichment and Sequencing of Targeted Genes The amplified DNA was captured using GenCap kidney disease capture kit (MyGenostics Inc, Beijing, China).The biotinylated 100 bp capture probes were designed to tile along the coding exons plus 50 bp flanking regions of all the genes. The capture experiment was conducted according to manufacturers protocol. Briefly, DNA library of 500ng was mixed with Buffer BL and GenCap gene panel probes (MyGenostics Inc), firstly. The mixture was heated at 95C for 5min, and then 65C for 5 min on a PCR machine. After that, 19 l of the 65C prewarmed Buffer HY (MyGenostics, MD, USA) was added into the mixture, and this mixture was held at 65C with PCR lid heat on for 16-24 hours for hybridization. 50 l of MyOne beads (Life Technology) was washed using 50 l of 1X binding buffer for 3 times, and then they were resuspended in 50 l of 1X binding buffer. The hybrid mixture. Then, beads were washed with WB1 buffer at room temperature for 15 minutes once, and WB3 buffer at 65C for 10 minutes three times. The bounded DNA was eluted with buffer, and amplified for 13 cycles using the following program: 95C for 4min s (1 cycle); 98C for 30 s, 65C for 30 s, 72C for 30 s (13 cycles); 72C for 5 min (1 cycle). The PCR product was purified using SPRI beads (Beckman Coulter) according to manufacturers protocol. The enrichment libraries were sequenced on Illumina HiSeq X ten sequencer for paired-reading of 150 bp.