Agilent SureSelect Human All Exon + UTR (v5): Sample HCI013_pdx_P3_XXX_WXS_10960X17
Library construction is performed using the Agilent Technologies SureSelect XT Reagent Kit, HSQ (cat # G9611A) as described below.  Briefly, genomic DNA (approximately 200 ng to 3 ug) is sheared in a 130 ul volume using a Covaris S2 Focused-ultrasonicator with the following settings: Intensity 5.0; Duty Cycle 10%25; Cycles per Burst 200; Treatment Time 6:00 minutes.  Sheared DNA is converted to blunt-ended fragments with 5'-phosphates and 3'-hydroxyl groups using a combination of enzymes that perform fill-in reactions and exhibit exonuclease activity.  An A-base is added to the blunt-ended DNA as a means to prepare the fragments for adapter ligation and to block concatamer formation during the ligation step.  Adapters containing a T-base overhang are ligated to the A-tailed DNA fragments.  Ligated fragments are PCR-amplified (6 cycles) and the amplified library is purified using Agencourt AMPure XP beads.  The concentration of the amplified library is measured on a NanoDrop spectrophotometer and an aliquot of the library is resolved on an Agilent 2200 Tape Station using a D1K (cat# 5067-5361 and 5067-5362)assay to define the size range.    Amplified libraries (750 ng) are hybridized with biotinilyated RNA baits from the Agilent Technologies SureSelect(XT) Human All Exon v5 plus UTRs kit  (5190-6213).  Library molecules hybridized to the SureSelect baits are purified using Dynabeads MyOne Streptavidin T1 (Life Technologies cat# 65601).  Index tags are added to the adapters of the captured library by pcr (10 cycles) using Agilent Herculase II Fusion DNA polymerase (600677) and primers from the Agilent Technologies SureSelect XT Reagent Kit and the amplified library is purified using Agencourt AMPure XP beads.  The concentration of the amplified, exome-enriched library is  measured using the Invitrogen Qubit dsDNA HS Assay (Q32851) and an aliquot of the library is resolved on an Agilent 2200 Tape Station using a D1K (cat# 5067-5361 and 5067-5362) or a High Sensitivity D1K (cat# 5067-5363 and 5067-5364) assay to define the size distribution of the sequencing library.  Libraries are adjusted to a concentration of approximately 5 nM and quantitative PCR is performed using the KapaBiosystems Kapa Library Quant Kit (cat# KK4824) to calculate the molarity of adapter ligated library molecules.  The concentration is further adjusted following qPCR to prepare the library for Illumina sequence analysis.