Targeted next-generation sequencing on ATM gene
Genomic DNA was extracted from peripheral blood leukocytes by standard methods. The qualified genomic DNA sample was randomly fragmented by ultrasonoscope (Covaris S2, Massachusetts, USA) and the size of the library fragments was mainly distributed between 200 to 250bp. Next, purified DNA was treated with T4 DNA polymerase, T4 phosphonucleotide kinase and the Klenow fragment of Escherichia coli DNA polymerase to fill 5' overhangs and remove 3' overhangs. Terminal A residues were added following the incubation with dATP and the Klenow 3'-5' exo-enzyme by standard Illumina protocols. Then adapters were ligated to both ends of the resulting fragments.  A custom Human capture array (Roche NimbleGen, Madison, WI) was designed to capture the fragments including exons, splice sites, and the adjacent intron sequences of the ATM gene, with subsequent sequencing performed with 90bp paired-end reads on a HiSeq2000 instrument (Illumina, San Diego, CA).