title: Whole exome sequencing of colorectal cancer cousin pairs.: Sample 10297X1_442_5
design: Library construction is performed using the Illumina TruSeq  DNA Sample Preparation Kit (cat# FC-121-2001, FC-121-2002) using the following methods.  Genomic DNA (approximately  10 ng to 2 ug) is sheared in a volume of 52.5 ul using a Covaris S2 Focused-ultrasocinicator with the following settings: Intensity 5.0; Duty cycle 10%25; Cycles per Burst 200; Treatment Time 120 seconds.  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.  Size selection of the blunt-ended DNA (200-450 bp average) is accomplished using bead-based methodologies.  An A-base is added to the blunt ends as a means to prepare the fragments for adapter ligation and 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 (2-10 cycles depending on quantity of input template) and the amplified library is purified by bead based methodologies.  The concentration of the lamplified ibrary 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 10 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.