Pair end library preparation kit

Genomic DNA was sheared using Covaris S series (Covaris, MS, USA). The sheared DNA was end-repaired, A-tailed, and ligated to pair-end adapters, in accordance with the manufacturer’s protocol (Pair End Library Preparation Kit, Illumina, San Diego, CA, USA). Adapter-ligated fragments were purified and dissolved in 30 μl of elution buffer, and 1 μl of the mixture was used as a template for 12 cycles of PCR amplification. The PCR product was gel-purified using the QIAquick Gel Extraction Kit (Qiagen). Library quality and concentration were determined using an Agilent 2100 BioAnalyzer (Agilent). Libraries were quantified using a SYBR green qPCR protocol on a LightCycler 480 (Roche, Indianapolis, IN, USA), in accordance with Illumina’s library quantification protocol. Based on the qPCR quantification, libraries were normalized to 2 nM, and then denatured using 0.1 N NaOH. Cluster amplification of denatured templates was performed in flow cells, in accordance with the manufacturer’s protocol (Illumina). Flow cells were paired-end sequenced on an Illumina HiSeq 2000 using HiSeq Sequencing kits. A base-calling pipeline (Sequencing Control Software (SCS), Illumina) was used to process the raw fluorescent images and the called sequences.

Genomic DNA was extracted from patient leukocytes by a MagAttract DNA Blood Midi Kit (Qiagen, Inc. Valencia, CA, USA) according to the manufacturer's protocol. The DNA quality was assessed using a Nanodrop spectrometer (Nanodrop Technologies, Wilmington, DE, USA). An absorbance 260/280 value greater than 1.7 was accepted for further analysis. Five micrograms of genomic DNA was sheared using a Covaris S series ultrasonicator (Covaris, Woburn, MA, USA). The fragments of sheared DNA were then end-repaired, A-tailed, ligated to pair-end adapters (Pair End Library Preparation Kit, Illumina, CA, USA), and amplified by PCR according to the manufacturer's protocol. The quality of the library and DNA concentration were determined using an Agilent 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA) and quantified using a SYBR green qPCR protocol on a LightCycler 480 (Roche, Indianapolis, IN, USA) according to Illumina's library quantification protocol. Paired-end sequencing (2 × 100 bp) was performed on an Illumina HiSeq 2000 by using HiSeq Sequencing kits