Ampure xp reagent beads

To prepare libraries for Whole Exome Sequencing, 100ng-1µg of genomic DNA was sheared with the Covaris S220 (Covaris, Woburn, MA, USA), followed by end-repair, 3’ end Adenylation and ligation with paired-end adaptors. Post ligation, 15µl of the purified libraries were PCR amplified, all the above steps were performed using the Agilent SureSelect^XT kit and every step was followed by DNA purification on a magnetic stand using AMPure XP Reagent beads (Beckman Coulter Genomics, Danvers, MA, USA). Afterward, size (approx 225-275 bp) and quantity (>800ng) were verified employing the Agilent Tapestation 2200 system followed by hybridization and probe capture using Exome SureSelect Human All Exon V6+UTR probes (Agilent). Dynabeads MyOne Streptavidin T1 magnetic beads (Thermofisher). Finally, Captured Libraries were amplified with 12 cycles of PCR using indexing primers containing 8-bp indices, followed by an amplification using AMPure XP beads (Beckman Coulter). Final libraries were checked for quality (each fragment size approx. 300-400 bp) and quantity using Agilent Tapestation 2200 system.

Fifty-five μl of Agencourt AMPure XP Reagent beads (Beckman Coulter, Brea, CA, USA) was added to 50 μl of isolated plasma DNA and the solution was mixed thoroughly. The solution was incubated for 5 minutes at room temperature. The tube was placed into a magnetic rack for 5 minutes and the supernatant was transferred into a new tube and 1.8 x volume of beads was added. The solution was mixed by pipetting up and down and incubated for 5 minutes at room temperature. The tube was placed into the magnetic rack for 5 minutes. The pellet was washed with 100 μl 70% ethanol and dried. The pellet was resuspended in 79 μl or 50 μl of Low TE solution and incubated for 5 minutes at room temperature. The tube was placed into the magnetic rack for 5 minutes and supernatant was collected and used in library preparation on Ion Torrent PGM and MiSeq, respectively. Five of the size selected DNA samples were used for Ion Torrent PGM and five for MiSeq library preparation.