7442 Nucleic Acids Research, 2009, Vol. 37, No. 22 the presence of a degenerate DNA sequence motif (THEMIS) in the mutation sites (11). The THEMIS motif {WKVnRRRnVWK: W = A/T, K = G/T, V = G/ A/C, R = purine (A/G); n = any nucleotide; total number of n = 0–2 nucleotides; the underline indicates the position of the mutated base}, was found signiﬁcantly more often than expected at the somatic mutation sites of both prostate and breast cancer tissue, when one mismatched was allowed (11). Here we utilize a PCR-free cloning/ sequencing approach to report much higher somatic mutation frequencies than previously measured in cancer tissue, mostly from intergenic regions across the tumor genome. MATERIALS AND METHODS Samples Prostatic adenocarcinoma patients used for the assessment of variation frequencies were of African American or Hispanic American background and were obtained from the Multiethnic Cohort (12). Patient age at diagnosis varied from 55 to 66 years and grading (Gleason score) varied from 6 to 7. One of the patients refused to provide blood DNA. For the assessment of heterogeneity we used Caucasian prostatic adenocarcinoma patients taken from a diﬀerent dataset (13). Patient age at diagnosis varied from 56- to 72-years old and stage varied from pT2a to pT3c (using the TNM staging system (14). DNA isolation Prostate tissue and blood were collected from each patient. Prostate tissue was microdissected manually. DNA was extracted from the prostate as in ref. (13) and from the blood with standard methods (15). Shotgun cloning and sequencing A 100–250 ng genomic DNA from matched lymphocytes, normal prostate and prostate tumor were digested with AluI (NEB, Beverly, MA, USA) and ligated into (SmaI) linearized, dephosphorylated pUC18. The plasmids were transformed into One Shot TOP10 Ultracompetent Escherichia coli cells (Invitrogen, Carlsbad, CA, USA) and recombinants were identiﬁed via blue-white screening. Plasmids were spin-column prepared (Qiagen, Valencia, CA, USA) and DNA sequenced (Applied Biosystems BigDye v3.1, Foster City, CA, USA) using M13F or M13R universal primers (IDT, Coralville, IA, USA). Sequencing reactions were aliquoted in 0.2% SDS, heated to 98 C for 5 min, prior to puriﬁcation by AutoSeq50 size exclusion chromatography (Amersham Biosciences, Pittsburg, PA, USA). Puriﬁed samples were then run on an ABI 3100 automated DNA sequencer. Genomic heterogeneity assessment A 50–100 ng of matched prostate tumor and blood DNAs were PCR ampliﬁed on a Robocycler thermal cycler (Stratagene, La Jolla, CA) using 2.5 U AmpliTaq Gold DNA polymerase (Roche), 1.5 mM MgCl2, 0.5 mM (CA)8RY oligo and 5% v/v DMSO, using the following PCR protocol: 15 min at 95 C for 1 cycle, followed by 40 cycles of 2 min 20 s at 95 C, 1 min 20 s at 48 C and 2 min 20 s at 72 C. PCR products were AluI-digested and then shotgun cloned and sequenced as described above. Variant identiﬁcation We sequenced 60–100 clones from each blood, tumor and normal prostate sample, bi-directionally. Vector sequences were removed, and each clone pair (forward and reverse sequence) was aligned using BLAST 2 SEQUENCES (www.ncbi.nlm.nih.gov/BLAST/). Mismatches were manually edited based on the sequence information from both strands (to remove obvious base-calling errors). This ﬁnal edited sequence output was then compared to the nr-database in Genbank using BLASTN (www. ncbi.nlm.nih.gov/BLAST/). Variations with the best match were conﬁrmed by blasting against the genomic reference sequence. Conﬁrmed variations were scored, unless they represented >5% of the total sequence, in which case the clone was rejected from the analysis (for further explanation see the Discussion section). All conﬁrmed variations were blasted against dbSNP build 129 (http://www.ncbi.nlm.nih.gov/SNP/snp_blast ByOrg.cgi), to identify known polymorphisms. Statistics Variation frequency. All statistical analyses comparing variation frequency for the three groups (blood, normal prostate and prostate tumor) were performed using SAS statistical software, version 9.1 (SAS Institute, Cary, NC, USA), and all results were tested at a 5% level of signiﬁcance. Data were summarized using descrip- tive statistics such as mean and standard deviations. The analysis of variance (ANOVA) method and Bonferroni multiple comparison test as a post hoc analysis were used for comparing means for these three frequency groups. THEMIS motif. Two-sided P-values were calculated for the prostate tumor and blood/normal prostate groups using the chi-squared test. RESULTS We measured the frequency of small-scale somatic alterations in sporadic prostate cancer by using a PCR- free cloning and sequencing method (Supplementary Figure S1). This technique was used to identify the number of variants present in randomly cloned genomic pieces of prostate tumor, normal prostate and blood DNAs from the same individual by ﬁrst sequencing each DNA fragment and then comparing the sequence to the nr (nonredundant)- and refseq_genomic-databases in Genbank (Supplementary Figure S1). Our prostate tumor DNA samples were obtained by microdissecting formalin ﬁxed tissue slides under the microscope, a tech- nique that results in minimum normal DNA contamina- tion of the tumor material, but which may cause an artiﬁcial increase of the mutation frequencies (18).