Agilent 2100 bioanalyzer 7500 chip

A total of 122 genes were targeted for a final capture size of 400 kb (SureSelectXT Custom 3–5.9 Mb, Agilent) (Castellanos, et al., unpublished data), according to Agilent’s SureSelect protocol for Illumina paired-end sequencing. Briefly, 3.0 μg of genomic DNA was sheared on a Covaris E210 instrument (Covaris, Woburn, Massachusetts). The fragment size (150–300 bp) and the quantity were confirmed with an Agilent 2100 Bioanalyzer 7500 chip. Fragmented DNA was end-repaired, adenylated and ligated to Agilent indexing-specific paired-end adaptors. The DNA with adaptor-modified ends was PCR amplified (6 cycles, Herculase II fusion DNA polymerase, Agilent) with SureSelect Primer and SureSelect Pre-capture Reverse PCR Primer, quality controlled on the DNA 7500 assay for the library size range of 250 to 450 bp and hybridized for 24 hours at 65 °C (Applied Biosystems 2720 Thermal Cycler). The hybridization mix was washed in the presence of magnetic beads (Dynabeads MyOne Streptavidin T1, Life Technologies, Carlsbad, California) and the eluate was PCR amplified (16 cycles) in order to add the index tags using SureSelectXT Indexes for Illumina. The final library size and concentration were determined on an Agilent 2100 Bioanalyzer 7500 chip.

The library preparation for capturing of selected DNA regions was performed according to the SureSelect XT Target Enrichment System protocol for Illumina paired-end sequencing (Agilent). In brief, 3 μg of genomic DNA was sheared on a Covaris™ E220 focused-ultrasonicator. Fragment size (150-200 bp) and quantity were confirmed with an Agilent 2100 Bioanalyzer 7500 chip. The fragmented DNA was end-repaired, adenylated and ligated to Agilent indexing-specific paired-end adaptors. The DNA with adaptor-modified ends was PCR amplified (6 cycles, Herculase II fusion DNA polymerase) with SureSelect primers, quality controlled using the DNA 7500 assay specific for a library size of 250–350 bp, and hybridized for 24 hr at 65°C. The hybridization mixture was washed in the presence of magnetic beads (Dynabeads MyOne Streptavidin T1, Life Technologies), and the eluate PCR amplified (16 cycles) to add index tags using SureSelectXT Indexes for Illumina. The final library size and concentration was determined using an Agilent 2100 Bioanalyzer 7500 chip and sequenced on an Illumina HiSeq 2000 platform with a paired-end run of 2 × 76 bp, following the manufacturer’s protocol. 36 sample libraries were loaded in three lanes of HiSeq 2000.