Materials and Methods. enzyme with a typical six base pair recognition site. Bgl II is one of many restriction

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1 Materials and Methods ROMA In our present studies we primarily use representations made with Bgl II, an enzyme with a typical six base pair recognition site. Bgl II is one of many restriction enzymes that satisfy these useful criteria: it is a robust enzyme, its cleavage site is not affected by CpG methylation, it leaves a four base overhang, and its cleavage sites have a reasonably uniform distribution in the human genome. After cleavage with Bgl II, we ligate adapters, and use the resulting product as a template for a PCR reaction. Because PCR selects small fragments, Bgl II representations are made up of the short Bgl II fragments, generally smaller than 1.2 kb, and we estimate that there are about 200,000 of them, comprising about 2.5% of the human genome, with an average spacing of 17kb. For validation of CNP data, we perform one additional experiment using Hind III representations and arrays of Hind III probes. In all of the experiments described herein, we comparatively hybridized representations prepared in parallel. In preliminary work, we found that noise created by variable yields of the PCR reactions was lowest if the DNA from two samples being compared were prepared at the same time, and representations prepared from the same concentration of template, using the same protocols, reagents and thermal-cycler. Probe design Oligonucleotide probes were designed as described previously (1). Briefly, we performed an in silico Bgl II digestion of the human genome by locating all Bgl II restriction sites within the April 2003 draft assembly and storing all sequences of Bgl II fragments that are between 200 and 1200 bp in length. Fragments were annotated with 1

2 the counts of their substituent, overlapping 15-mers and 21-mers using the "mer-engine" constructed from the same draft assembly. For each fragment, the following attributes were determined for every substituent, overlapping 70-mer: maximum 21-mer count, arithmetic mean of 15-mer counts, percent GC content, the quantity of each base, and the longest run of any single base. All 70-mer probes that possess any of the following characteristics were eliminated: maximum 21-mer count >1, GC content <30% or >70%, a run of A/Ts >6 bases, a run of G/Cs >4 bases. From the remaining set of 70-mers, the one (or more) that has a GC/AT proportionality closest to that of the genome as a whole as well as a minimal mean 15-mer count were selected. As a final check for overall uniqueness, the optimal probes for each fragment were compared with the entire genome using BLAST (default parameters were used with the exception of filtration of low complexity sequence, which was not performed). Any probe found to have any degree of homology along 50% or more of its length to any sequence other than itself was eliminated. Oligonucleotide probes were synthesized in arrays on a glass surface using mirror directed laser photochemistry (Nimblegen). A major advantage of this fomat is that the cost of testing several different sets of probes is no more than the cost ordering multiple chips of a single design. Therefore, we tested 700,000 unique 70-nt probes predicted to be complementary to small BglII fragments, arrayed on eight chips. These were hybridized with standard Bgl II and EcoR I-depleted Bgl II representations, and we picked the 85,000 with the most intense signal when hybridized to a single normal human DNA, SKN1. These 85,000 were then arrayed on a single chip. The 85K photoprint 2

3 arrays used here were a gift of NimbleGen Systems Inc. (Iceland), or purchased and hybridized in our Iceland facility, LindGen EHF. Sample DNAs DNA samples from normal individuals were obtained from a collection maintained by Columbia Genome Center, Columbia University (Conrad Gilliam), and DNAs and cell lines were obtained from Coriell Cell Repository (NIGMS). Sample and experiment descriptions are provided in table S1. Samples of sperm and blood from donors 1-8 were obtained for this study through informed consent. DNA was purified from whole blood using the Puregene blood DNA isolation Kit (Gentra Systems). DNA was purified from sperm using the QIAamp tissue kit (Qiagen). Sample and experiment descriptions are provided in Table S1. Sample preparation, microarray hybridization and image analysis The preparation of genomic representations, labeling and hybridization were performed as described previously (2). Briefly, the complexity of the samples was reduced by making Bgl II genomic representations, consisting of small ( bp) fragments amplified by adaptor-mediated PCR of genomic DNA (3). For each experiment, two different samples were prepared in parallel. DNA samples (10 µg) were then labeled differentially with Cy5-dCTP or Cy3-dCTP using Amersham-Pharmacia Megaprime labeling Kit, and hybridized in comparison to each other. Each experiment was hybridized in duplicate, where in one replicate, the Cy5 and Cy3 dyes were swapped (i.e. color reversal ). Hybridizations consisted of 25 µl of hybridization solution (50% formamide, 5x SSC, and 0.1%SDS) and 10 µl of labeled DNA. Samples were denatured 3

4 in an MJ Research Tetrad at 95 C for 5 min, and then pre-annealed at 37 C for 30 min. This solution was then applied to the microarray and hybridized under a coverslip at 42 C for 14 to 16 h. After hybridization, slides were washed 1 min in 0.2% SDS/0.2x SSC, 30 sec in 0.2x SSC, and 30 sec in 0.05x SSC. Slides were dried by centrifugation and scanned immediately. An Axon GenePix 4000B scanner was used setting the pixel size to 5 µm. GenePix Pro 4.0 software was used for quantitation of intensity for the arrays. Data processing Array data were imported into S-PLUS for further analysis. Measured intensities without background subtraction were used to calculate ratios. Data were normalized using an intensity-based lowess curve fitting algorithm similar to that described in Yang et al. (4). Log ratio values obtained from color reversal experiments were averaged and displayed as presented in the figures. Copy number polymorphisms (CNPs) were identified based on probe ratios using a Hidden Markov Model (HMM, as described in the text). Locus Link annotations that mapped within CNPs were identified based on the genomic coordinates of genes from UCSC GoldenPath (excluding genes that mapped to unassembled random contigs). Locus Link IDs were downloaded from the ftp site of NCBI, ftp://ftp.ncbi.nih.gov/refseq/locuslink/. RefSeq accession numbers and genomic coordinates (April, 2003 freeze) were downloaded from the UCSC GoldenPath (URL: Locus Link IDs, RefSeq IDs and genome coordinates were joined in a MySQL database server using SQL query 4

5 language. The complete list of Locus Link genome annotations used here contained 15,628 unique Locus Link identifiers consisting of 18,802 RefSeq genes. Hidden Markov Model program for identifying CNPs Microarray hybridization data were analyzed using a Hidden Markov Model (HMM) that was designed to distinguish differences in DNA copy number from other variation in probe ratios due to experimental noise and sequence polymorphisms. The HMM assumes that, in a comparison of two individuals, most intervals of the genome are equal in copy number, in the state we designate as 0. Some intervals of the genome may differ in copy number by discrete constant ratios, and it is assumed that all CNP states enter and leave through state 0. There are six experimentally distinguishable CNP states: ratios of 3:2 and 4:3 are designated as state +1 ; ratios of 4:2 and 2:1 are designated as state +2 ; any ratios >2:1 are designated as state +3 ; and the inverse of these ratios are designated as states -1, -2 and -3, respectively. The initial parameters of the HMM (means and standard deviations of states) were derived using the expectation maximization (EM) algorithm, and the transition probabilities were initialized by a nonparametric clustering of outliers. In the present study, a CNP is defined as the interval where the most probable path defined by the HMM is a CNP state. The statistical measure of confidence given to a CNP is the probability that the interval is not in the ground state. Lastly, the majority of single- and double-probe differences can be accounted for by sequence polymorphisms within one of the restriction sites of the Bgl II fragment (RFPs) (5). Hence, CNP intervals of less than 3 probes were disregarded. In addition, in order to eliminate all possible CNPs that could result from 2 independent sequence 5

6 polymorphisms, we also filtered out longer intervals that could potentially be caused by 2 SNPs. This may occur when two of the Bgl II fragments within a short CNP interval share a single restriction site between them. If this single Bgl II site is abolished by a sequence polymorphism, a 2-probe event results from a single SNP. It is thus feasible for a 3-probe event to result from 2 SNPs (one SNP affecting 2 probes and the other affecting one probe). It is also possible for a 4-probe event to result from 2 SNPs if the interval consists of 2 pairs of neighboring Bgl II fragments. We examined our data using the above criteria, we determined that eight segments met one of the above criteria, and these were subsequently removed from the analysis. Fluorescence in situ hybridization For analysis of CNP32, Bacterial Artificial Chromosome plasmid DNA (CTD- 2314K17) was used as probe. Metaphase spreads were prepared from cell lines using published procedures (6), and methods for probe preparation, hybridization, washing, detection with FITC, fluorescent banding, and analysis are described elsewhere (6) with the exception that approximately 250 ng of biotinylated BAC DNA was applied to each slide. The remaining FISH analysis was done using interphase cells, and probe DNA was amplified from specific genomic regions by PCR. Based on the human genome sequence, primers (1-2 Kb in length) were designed from the repeat-masked sequence of each CNP interval, and limited to an interval no larger than 100 Kb. For each probe, a total of different fragments were amplified, then pooled, and purified by ethanol precipitation. Probe DNA was then labeled by nick translation with SpectrumOrange or SpectrumGreen (Vysis Inc.,Downers Grove, IL). All hybridizations contained one 6

7 probe complementary to the CNP interval. Wherever necessary, a control probe, labeled in the opposite color, was also included in the same hybridization in order to confirm that cells were diploid (ploidy control). In one instance, the ploidy control used was a probe designed to hybridize with the MSF gene, which did not overlap with any CNP interval (Fig 3, panel A). In all other cases, a probe complementary to one of the CNP intervals served as a ploidy control because the individual being examined was not variant at this specific genomic location. Denaturation of probe and target DNA was performed at 90 C for 5 minutes, followed by hybridization in a humidity chamber at 47 C over night. The cover glasses were then removed and the slides were washed in 2x SSC for 10 minutes at 65 C, and slides were dehydrated in graded alcohol. The slides were mounted with anti fade mounting medium containing DAPI (4, 6-diamino-2-phenylindole, Vectashield) as a counter-stain for the nuclei. Cover slips were applied and sealed with transparent nail polish. Evaluation of signals was carried out in an epifluorescence microscope. Selected cells were photographed in the multidimensional imaging system Deltavision. In experiments using interphase cells and only a single probe for the test locus (no ploidy control), we screened cell nuclei in order to obtain a conclusive estimate of copy number. The images presented in Figure S2 are typical representative copy number. Regarding deletions the situation is as follows: In a normal diploid fibroblast we see 2 dots in % of the nuclei and 1 dot in % of the nuclei (due to a FISH artifact). Occasionally, in less than 1 % of the nuclei, we see no dot at all, due to the same artifact. In a fibroblast with one allele deleted we see 1 dot in % of the nuclei and no dot in % of the nuclei. In the fibroblast population we have two other sources of variation. (1) About % of the cells are tetraploid, and there we see 4 or 3 7

8 dots (occasionally 2 due to the above mentioned FISH-artifact). (2) About 20 % of the cells are in S- or G2-phase of the cell cycle. These cells also have 4 or 3 dots, however in these cases the dots are in pairs due to DNA-replication (sister chromatids). The above criteria are sufficient for determining the presence of a deletion or duplication, provided that there is a good FISH signal, which was the case for all of the genes examined. Determining germline and somatic variation in copy number Observed differences in the copy number of genome segments between samples from two individuals could reflect germline differences or somatic variation. Therefore, for our initial experiments, we sampled two tissues from each of four donors (sperm and whole blood). For each donor, DNA from both tissues were compared to a standard reference DNA (SKN1) from an unrelated individual, and blood and sperm from the same donor were compared to each other. SKN1 reference DNA was derived from a low passage non-immortalized culture of fibroblasts cells from a normal male (2). A comparison of SKN1 fibroblast DNA and sperm from the same individual showed no detectable somatic variation (7). Thus, when sperm, blood and SKN1 are analyzed in all three pair-wise combinations, a germline CNP should be apparent in both donor tissues in comparison to SKN1; whereas, a copy number difference arising somatically should be apparent as a difference between tissues. In the above comparisons, only two somatic mutations were observed, both in the same donor, and both at T-cell receptor loci (7p14 and 14q11). Figure S1 illustrates examples of a germline CNP and a somatic variation from one donor when he is compared to SKN1. The microarray ratio data are plotted for each pair-wise comparison, 8

9 and the segments of altered copy number predicted by the HMM (in the blood vs. SKN1 experiment) are represented by a solid grey line. The germ-line polymorphism (panel A) is at 6p21, spans >230 Kb, and contains several genes including pancreatic colipase, CLPS (CNP34, Table 1). The somatic mutation (CNP85, panel B) spans most of the T- cell receptor alpha locus. CNP85 may reflect normal VDJ recombination of T-cells, a major component of blood. Our study was expanded further using the two most commonly available sources of genetic material: whole blood and EBV-immortalized lymphoblastoid cell lines (LCLs) (see experiment descriptions, table S1). It is not known if LCLs develop lesions that go undetected by routine cytogenetic examination, which could be misinterpreted as CNPs. Therefore, in a manner similar to our analysis of blood and sperm, we performed a triangulation of blood and LCLs from four donors and SKN1. Our results indicated that copy number differences between whole blood and LCLs were located at the immunoglobulin IGH, IGL and IGK gene clusters (CNPs 13, 68 and 86), which reflect normal VDJ-type recombination of lymphoid cells. Therefore, the use of LCLs in this study is not problematic. Nonetheless, the possibility of somatic mutations or rare artifacts of cell culture cannot be ruled out. Therefore, a CNP was considered to be germline if it was observed twice in this study (i.e. observed in multiple individuals or in multiple tissues of the same individual). Experimental validation of and limits in detecting copy number polymorphisms Using the methods described below, independent experimental validation of ROMA data was sought (Fig. 2, Fig. S1 and Table S1). 9

10 Lymphoblastoid cell lines were obtained from several individuals in this study, and examined by fluorescence in situ hybridization (FISH). Twelve probes were designed to hybridize with twelve different CNP intervals, as described above. Nine of twelve CNPs were unambiguously confirmed by cytogenetic analysis (Fig. 3 and Fig. S1). Five CNPs were found to be hemizygous deletions, and four were duplications. Additional validation of CNPs was obtained by microarray analysis of genomic representations made using a different restriction enzyme. Where probe coverage of a CNP interval is sufficient (> 3 probes/cnp), a CNP should be apparent in two different representations, for example Bgl II and Hind III. One pair of individuals was analyzed by Bgl II-ROMA (experiment JA437, table S1), and the same pair of individuals was analyzed using Hind III representations and arrays of Hind III probes (JT393). Of all CNPs observed by Hind III-ROMA, eight CNP intervals also contained sufficient coverage by Bgl II probes (> 3 probes). All but one of these (CNP87) were also detected by Bgl II-ROMA. In the interval of CNP87, 5 of 6 Bgl II probes reported a ratio < 0.9; however, the HMM prediction did not determine this interval to be a CNP in this experiment. Due to differences in the genomic distribution of Hind III probes, some unique CNPs were identified. Eleven novel CNPs were detected by Hind III-ROMA for which the coverage by Bgl II probes was not sufficient. Estimating our rate of error In order to estimate the rate of false-positive and false-negative results in our data, we examined the reproducibility of CNPs in experiments that analyzed blood and LCLs from the same donor and SKN1 in all three pair-wise combinations. A CNP that was detected in only one of the above comparisons indicated an error, either a false-positive 10

11 or false-negative. If a CNP was detected in only one of the three pair-wise comparisons yet was detected in a different individual in our study, the failure to detect it in two pairwise comparisons was considered to indicate a false-negative. Otherwise, it was considered to be a false-positive, a slight overestimate. Based on these criteria, using our present HMM analysis and technology, we fail to detect 33% of the CNPs that could be detected by BglII ROMA, and 6 percent of those we do detect are false-positives (Table S4). Given the criteria above, we expect some false-negatives to be misclassified as false-positive. A high proportion of CNPs are rare, and it is likely that some of the results classified as "false positive" are in fact rare CNPs, and would not be observed twice in 20 donors. Our failure to detect such a CNP in both replicate experiments could be due to a false negative in one experiment (not a false positive). Therefore, our falsepositive rate is probably a slight overestimate, and our false-negative rate, an underestimate. 11

12 Figure legends Figure S1. Examples of germline and somatic copy number variation identified by ROMA. Sperm and blood of from the same donor and reference DNA (SKN1) were analyzed by ROMA in three pair-wise comparisons: sperm vs SKN1 (blue), blood vs SKN1 (red) and blood vs sperm (green). The Y-axis is the geometric mean (from color reversal experiments), on a log scale, of the ratio of intensities of probes from selected genomic regions, in chromosome order. The horizontal (grey) line represents the median ratio of each interval of the HMM most probable path for the blood vs SKN1 data. (A) A copy number loss at 6p21 is apparent only in the sperm/skn1 and blood/skn1 experiments, and (B) a copy number loss at 14q11 arising somatically in blood is apparent only in the blood/skn1 and blood/sperm comparisons. Figure S2. Validation of ROMA results by fluorescence in situ hybridization (FISH). Panels to the left present CNPs identified by ROMA (A, C, E, H, and K). Listed in these panels are the CNP ID#, experiment name and the name of one gene located entirely with the interval. Panels to the right present cytogenetic analysis of interphase cells from one or both individuals using probes that target the same CNP intervals. Each hybridization included a control probe (labeled in the opposite color) in order to confirm that cells were diploid. (B) CNP2 probe (red) in SKN1 cells; (D) CNP13 probe (green) in SKN1 cells; (F) CNP54 probe (green) in GM11524 cells and (G) in GM11322 cells; (I) CNP22 probe (green) in SKN1 cells; (K) CNP* probe (red) in GM12547 cells. Two different alleles of the CNP* polymorphism can be seen GM12547 cells, one allele gives a single signal, and a second allele gives a cluster of signals (indicated by an arrow). According to protocol, CNP* was omitted from table S2 because it was an interval of < 3 probes. 12

13 Fig. S1 13

14 Fig. S2 14

15 Table S1. Summary of experiments and research subjects Experiment Format Sample ID Sample description donor Sample source Reference ID Sperm/blood 3-way JT485 Bgl II B0002 blood DNA 2 This study SKN1 JT487 Bgl II SP0002 sperm DNA 2 This study SKN1 JT489 Bgl II B0002 blood DNA 2 This study SP0002 JT491 Bgl II B0004 blood DNA 4 This study SKN1 JT493 Bgl II SP0004 sperm DNA 4 This study SKN1 JT495 Bgl II B0004 blood DNA 4 This study SP0004 JBH015 Bgl II SP0008 blood DNA 8 This study SKN1 JBH017 Bgl II B0008A sperm DNA 8 This study SKN1 JBH019 Bgl II SP0008 blood DNA 8 This study B0008A JT497 Bgl II B0008B blood DNA 8 This study SKN1 JT499 Bgl II SP0008 sperm DNA 8 This study SKN1 JT501 Bgl II B0008B blood DNA 8 This study SP0008 JT503 Bgl II B0009 blood DNA 9 This study SKN1 JT505 Bgl II SP0009 sperm DNA 9 This study SKN1 JT507 Bgl II B0009 blood DNA 9 This study SP0009 LCL/blood 3-way JT509 Bgl II CG0002 blood DNA 5738 C. Gilliam SKN1 JT511 Bgl II CG0003 LCL DNA 5738 C. Gilliam SKN1 JT513 Bgl II CG0002 blood DNA 5738 C. Gilliam CG0003 JT515 Bgl II CG0004 blood DNA 5179 C. Gilliam SKN1 JT517 Bgl II CG0005 LCL DNA 5179 C. Gilliam SKN1 JT519 Bgl II CG0004 blood DNA 5179 C. Gilliam CG0005 JT521 Bgl II CG0008 blood DNA 5256 C. Gilliam SKN1 JT523 Bgl II CG0009 LCL DNA 5256 C. Gilliam SKN1 JT525 Bgl II CG0008 blood DNA 5256 C. Gilliam CG0009 JT533 Bgl II CG00014 blood DNA 5737 C. Gilliam SKN1 JT535 Bgl II CG00015 LCL DNA 5737 C. Gilliam SKN1 JT537 Bgl II CG00014 blood DNA 5737 C. Gilliam CG00015 Additional Donors JA437 Bgl II NA10470 LCL DNA Biaka Pygmy Coriell SKN1 JA440 Bgl II NA11524 LCL DNA Druze Coriell NA11322 JA442 Bgl II NA10540 LCL DNA Melanesian Coriell NA11589 JA542 Bgl II NA12547 LCL DNA French Father Coriell SKN1 JA544 Bgl II NA12548 LCL DNA French Mother Coriell SKN1 JA546 Bgl II NA12550 LCL DNA French Daughter 1 Coriell SKN1 JA548 Bgl II NA12552 LCL DNA French Son 2 Coriell SKN1 JT257 Bgl II NA13181 LCL DNA Venezualan Coriell SKN1 JT259 Bgl II NA11376 LCL DNA Cambodian Coriell SKN1 JT261 Bgl II NA10975 LCL DNA Mayan Coriell SKN1 JT263 Bgl II NA10495 LCL DNA Mbuti Pygmy Coriell SKN1 JT267 Bgl II NA10540 LCL DNA Melanesian Coriell SKN1 JT393 Hind III NA10470 LCL DNA Biaka Pygmy Coriell SKN1 JT527 Bgl II CG10 blood DNA 5264 C. Gilliam SKN1 15

16 Table S2. CNPs identified by ROMA. All CNPs listed here were identified by the HMM with a probability > For each CNP, we list the start position and length of the minimal interval determined by the HMM (In one case, CNP36, the interval spanned a centromere, and therefore was divided into two intervals, a and b). The status of a CNP was determined to be somatic (Som) if it occurred as a difference between tissues of the same individual. The status of CNP was determined to be germline (Germ) if it occurred in multiple tissues of the same individual or multiple individuals. One interval contained germline and somatic variants (Germ*). Where indicated, experimental confirmation of CNPs was obtained by (F) FISH or (H) analysis of Hind III and Bgl II representations. No estimates of minor allele occurences were made for CNPs identified only by Hind III- ROMA (Hind). Some of the CNPs listed were determined to be germline based on their occurrence in parents and progeny of CEPH pedigree 66; however, progeny are not included in the occurrence rate. 16

17 Table S2. # of Copy # CNP probes Change Chr Band CNP start Length Status Confirm Total Occurence Occurence of Minor Allele 1 3 Gain 1 p Germ Loss 1 p Germ F Gain 1 p Loss 1 q Loss 1 q Loss 1 q Germ Loss 1 q Hind 9 8 Loss 1 q Germ Loss 2 p Loss 2 p Germ Gain 2 p Gain 2 p Germ* F,H Gain 2 q Germ Loss 2 q F Loss 2 q Germ Loss 2 q Gain 3 q Gain 4 p Germ Loss 4 p Loss 4 p Germ F Gain 4 q Germ F,H Loss 4 q Hind Loss 4 q Gain 5 p Germ Loss 5 p Loss 5 p Hind 28 4 Gain 5 p Hind 29 5 Loss 5 q Germ H Gain 6 p Germ F,H Loss 6 p Germ Loss 6 p Germ F a 17 Loss 6 p b 17 Loss 6 q Loss 6 q Germ Loss 6 q Germ Loss 6 q Loss 7 p Germ Loss 7 q Germ Gain 8 p Gain 8 p Germ Gain 8 p Germ Loss 8 p Hind 47 3 Gain 8 q Loss 8 q

18 # of Copy # Total Occurence of CNP probes Change Chr Band CNP start Length Status Confirm Occurence Minor Allele 49 8 Loss 8 q Gain 9 p Germ Gain 9 p Germ Loss 9 q Loss 9 q Germ Loss 9 q F Loss 9 q Germ Gain 10 q F Loss 11 q Loss 11 q Loss 12 p Loss 12 q Loss 13 q Loss 13 q Loss 13 q Germ Loss 13 q Germ Gain 14 q Germ H Gain 14 q Som H Loss 15 q Germ H Gain 15 q Gain 15 q Gain 15 q Loss 15 q Germ F Gain 16 p Germ Gain 16 p Germ Loss 16 q Germ Gain 17 q Hind 79 9 Gain 17 q Germ Gain 17 q Hind 81 5 Loss 19 p Hind 82 7 Loss 21 q Loss 22 q Hind 84 3 Loss 7 p Som Loss 14 q Som Loss 22 q Som Loss 12 p Hind 88 9 Gain 22 q Hind 18

19 Table S3. Estimation of the rate of false positive and false-negative results. Blood DNA (replicate A) and LCL DNA (replicate B) from each of four donors were analyzed by ROMA in comparison to SKN1. In addition, blood and LCL from the same donor were compared to each other. Reproducible CNPs correspond to the CNPs that occurred in two of the above comparisons (the number of reproducible CNPs may differ between replicates A and B because of somatic differences). CNPs that were not reproducible were compared to the cumulative results of this study. CNPs that were observed in other individuals were considered to be false-negatives, and CNPs that were not observed in any other experiment were considered to be false-positives. Experiment Donor Replicate name # Observed Reproducible False - False A JT B JT A JT B JT A JT B JT A JT B JT Total = Percent =

20 Table S4. Estimates of the total number of CNPs in 20 donors. Listed are the number of observed Bgl II CNPs of a given length (those with an occurrence > 1 are shown separately). Note, the Bgl II CNPs shown here do not include CNPs < 25 kb in length. Probability corresponds to the probability that an interval of a given length will contain 4 probes, and probabilities were determined based on the poisson distribution of 85,000 probes in the genome. The expected total was calculated based on all CNPs. The combined length of CNPs (Total Mb) was calculated based on the median length of the interval. For intervals with a probability of one, no estimation was necessary; therefore, we list the actual combined length instead of the estimated length. kb CNPs Occurrence >1 All CNPs Probability Expected Total Total Mb Total =

21 Table S5. Gene content of CNPs. All locus link annotations that map within CNPs (excluding somatic mutations) are listed below. CNP Locus Gene Link ID Symbol Gene Name Position Change in Copy # melanoma antigen 1p36.2 Gain RHCE Rhesus blood group, CcEe antigen 1p36.1 Loss RHD Rhesus blood group, D antigen 1p36.1 Loss small membrane protein 1 1p36.1 Loss melanoma antigen 1q21 Loss PDE4DIP phosphodiesterase 4D interacting prot. 1q21 Loss hypothetical protein FLJ q21 Loss FCGR2B Fc fragment of IgG, low affinity 1q23 Loss hypothetical protein MGC4677 2p11 Gain hypothetical protein FLJ q14 Loss hypothetical protein FLJ q14 Loss RAB6C member RAS oncogene family 2q14 Loss CPS1 carbamoyl-phosphate synthetase 2q34 Loss GBA3 glucosidase, beta, acid 3 (cyto 4p15.3 Loss ATOH1 atonal homolog 1 (Drosophila) 4q22 Gain GRID2 glutamate receptor, ionotropic, 4q22 Gain TLL1 tolloid-like 1 4q32. Loss ZDHHC11 zinc finger, DHHC domain containing 5p15.3 Gain CDK7 cyclin-dependent kinase 7 5q13 Loss GTF2H2 general transcription factor II 5q13. Loss BIRC1 baculoviral IAP repeat-containing prot. 5q13 Loss OCLN occludin 5q13 Loss RAD17 RAD17 homolog (S. pombe) 5q13 Loss SMN1 survival of motor neuron 1 5q13 Loss SMN2 survival of motor neuron 2 5q13 Loss TAF9 TAF9 RNA polymerase II 5q13 Loss SERF1A small EDRK-rich factor 1A 5q13 Loss SMA3 5q13 Loss SERF1B small EDRK-rich factor 1B 5q13 Loss hypothetical protein FLJ q13 Loss Kenae 5q13 Loss IRF4 interferon regulatory factor 4 6p25 Gain DUSP22 dual specificity phosphatase 22 6p25 Gain CLPS colipase, pancreatic 6p21.3 Loss FKBP5 FK506 binding protein 5 6p21.3 Loss C6orf81 chromosome 6 open reading frame 6p21.3 Loss DGKB diacylglycerol kinase, beta 90k 7p21 Loss ETV1 ets variant gene 1 7p21 Loss MYOM2 myomesin (M-protein) 2, 165kDa 8p23 Gain CSMD1 CUB and Sushi multiple domains 8p23 Gain 21

22 Locus Gene CNP Link ID Symbol Gene Name Position Change in Copy # DEFA5 defensin, alpha 5 8p23 Gain DEFB4 defensin, beta 4 8p23 Gain SPAG11 sperm associated antigen 11 8p23 Gain DEFB103 defensin, beta 103 8p23 Gain DEFB104 defensin, beta 104 8p23 Gain hypothetical protein MGC p23 Loss COH1 Cohen syndrome 1 8q22 Loss EXT1 exostoses (multiple) 1 8q24.1 Loss DKFZP572C163 protein 9p11 Gain cell recognition molecule CASPR 9p11 Gain hypothetical protein DKFZp434A1 9p11 Gain TLE1 transducin-like enhancer of split1 9q21.3 Loss PPYR1 pancreatic polypeptide receptor 10q11.2 Gain KIAA0514 gene product 10q11.2 Gain OR5I1 olfactory receptor, family 5 11q11 Loss TRIM48 tripartite motif-containing 48 11q11 Loss TRIM51 tripartite motif-containing 51 11q11 Loss seven transmembrane helix recept. 11q11 Loss USP15 ubiquitin specific protease 15 12q14 Loss CLYBL citrate lyase beta like 13q32 Loss KIAA0125 gene product 14q32.3 Gain C14orf110 chromosome 14 open reading fram 14q32.3 Gain CHRFAM7A CHRNA7 (cholinergic receptor) 15q13 Gain CHRNA7 cholinergic receptor, nicotinic acid 15q13 Gain TP53TG3 protein 16p11 Gain TBC1D3 TBC1 domain family, member 3 17q12 Gain NSF N-ethylmaleimide-sensitive factor 17q21.3 Gain ARF protein 17q21.3 Gain NPEPPS aminopeptidase puromycin sensit 17q21.3 Gain NCAM2 neural cell adhesion molecule 2 21q21 Loss 22

23 References and Notes 1. J. Healy, E. E. Thomas, J. T. Schwartz, M. Wigler, Genome Res (Sep 15, 2003). 2. R. Lucito et al., Genome Res 13, (Oct, 2003). 3. N. Lisitsyn, M. Wigler, Science 259, (Feb 12, 1993). 4. Y. H. Yang et al., Nucleic Acids Res 30, e15 (Feb 15, 2002). 5. The expected frequency of SNPs is 0.1 %. Therefore, the probability of a SNP occurring in the restriction sites flanking a Bgl II fragment is 1.2 %. The observed frequency of single-probe events in our data is 1.5 % per haploid genome; therefore, the majority of these can be accounted for by SNPs. Some RFPs have been confirmed by amplifying specific restriction sites by PCR and digesting the fragments with Bgl II 6. B. J. Trask, in Genome Analysis: A laboratory manual B. Birren et al., Eds. (Cold Spring Harbor Laboratory Press, USA, 1999), vol. 4, pp J. Sebat, J. Troge, M. Wigler, Data not shown 8. W. J. Ewens, J. Roy. Stat. Soc. 25, (1963). 9. M. Kimura, J. F. Crow, Genetics 49, (Apr, 1964). 10. L. Kruglyak, D. A. Nickerson, Nat Genet 27, (Mar, 2001). 23