Profile of somatic mutations in four CLL genomes.
Four patients with CLL, who had given informed consent for sample collection and analysis, were studied. Tumour samples were obtained before treatment and tumour cells were separated from non-tumour cells by immunomagnetic depletion of T cells, natural killer cells, monocytes and granulocytes (Supplementary Information). Tumour cell purity was ≥98% as assessed by flow cytometry. Normal blood cells from the same patient were obtained after treatment, resulting in no detectable, or less than 0.05%, tumour cell contamination, as assessed by flow cytometry. Additional samples from 363 patients were obtained for clinical validation. Protocols for long-insert and short-insert library construction and for massively parallel paired-end sequencing have been described elsewhere (ref. 25 and Supplementary Information). Genotyping and copy number analysis were performed using the Affymetrix SNP6.0, Agilent 1M and Illumina OmniQuad arrays on the same cases used for whole-genome sequencing. For the validation of candidate genes in a set of 169 additional CLL patients, we used a combination of PCR amplification and Illumina sequencing in pooled samples, resulting in efficient identification of germline and somatic mutations (Supplementary Information). Sequencing data were aligned to the human reference genome (GRCh37) using Burrows–Wheeler alignment (BWA)26 and somatic substitutions were identified using Sidrón, a probabilistic binomial model that uses genotyping data to calibrate sequencing error per sample. Functional analyses of the identified mutations were performed using cryopreserved primary tumour cells. For gene expression analysis, RNA was purified from tumour cells and analysed using the HU133 plus 2.0 GeneChip (Affymetrix). For immunoprecipitation and western blotting, CLL cell extracts were prepared and detected using the indicated antibodies (Supplementary Information). For Toll-like receptor stimulation of CLL cells, the Human TLR1–9 agonist kit (InvivoGen) was used.