Exome sequencing of Homo sapiens: 3 - year - old male child
6.2.1 Sample Fragmentation For genomic DNA, calculate the loading amount per library (recommended 100 ng) based on qubit value, then adjust volume with water. ctDNA doesn't need fragmentation. Add prepared fragmentation reagent to the diluted library. Run the fragmentation program on a PCR instrument (4, 25 for 25 min, then 4). Add reagents on ice and set PCR hot - lid at 47. After reaction, place tube on ice and add Stop Solution. Add XP Beads, vortex - mix, and incubate at room temperature. Centrifuge, place on magnetic rack, and discard supernatant. Wash with 80% ethanol twice, dry at room temperature. Add NFWater, resuspend beads, incubate, then transfer supernatant to a new tube. Store unused reagents at - 20 and beads at 4. Fragmented DNA can be stored at 4 overnight or - 20 long - term. 6.2.2 End Repair & A - Tailing Thaw relevant buffers and enzymes on ice, mix, and centrifuge. Prepare reaction system in a cold PCR tube. Mix and centrifuge the reaction. Run the reaction program (20, 65, then 10 hold) on the PCR instrument. Store unused reagents at - 20. 6.2.3 Adapter Ligation Thaw ligation - related reagents on ice, mix, and centrifuge. Prepare adapter ligation reaction system using the previous step's product. Mix and centrifuge the reaction. Run the reaction program (20, then 4 hold) on the PCR instrument. No hot - lid needed, and reaction time can be extended (max 3 hours). Store unused reagents at - 20. 6.2.4 Purification Transfer the reaction to a tube with equilibrated magnetic beads, mix, and incubate. Centrifuge, place on magnetic rack, and discard supernatant. Wash with 80% ethanol twice, dry at room temperature. Add NFWater, resuspend beads, incubate, then transfer supernatant to a new tube. Store unused reagents at - 20 and beads at 4.