Wu et al., Determination of genetic identity in therapeutic chimeric states. We used two approaches for identifying potentially suitable deletion loci

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1 SUPPLEMENTARY METHODS AND DATA General strategy for identifying deletion loci We used two approaches for identifying potentially suitable deletion loci for PDP-FISH analysis. In the first approach, we used a published database by based on computational SNP analysis to explore potential loci (Supplementary Fig. 1, and as described also in the main text) 1. As a second approach, additional loci were identified based on our observations of recurrent deletion loci using array CGH (Supplementary Fig. 2). Sensitivity of polymorphic deletion probes To determine the validity and utility of these autosomal chromosomal deletion probes for genetically discriminating two individuals in chimeric states, we performed controlled mixing studies of two HapMap individuals with defined deletions and who were gender mismatched (male NA10838 is Chr2p PDP (+/+) and Chr8p PDP (+/ ) and female NA10859 is Chr2p PDP ( / ) and Chr8p PDP (+/+)). Gender mismatching allowed us to use traditional sex chromosome based FISH analysis as an internal control for chimerism assessment. Assessment of the extent of chimerism in these idealized mixing studies using FISH for the sex chromosomes was reliably and accurately reproduced by FISH using PDPs targeting the autosomal deletion loci on chromosomes 2p and 8p, (linear regression, slope = 0.995, R 2 = 0.997, Supplementary Fig. 3). To determine the sensitivity of PDP-FISH for chimerism assessment as compared to standard PCR-based short tandem repeat (STR) analysis, we 1

2 serially diluted DNA from male (NA10838) cells into female (NA10859) cells at varying ratios and assessed chimerism using both methods. As shown, our current routine clinical chimerism assay that uses PCR-based STR analysis is sensitive to ~1% chimerism (Supplementary Fig. 4a). While we can detect the male NA10838 cells by FISH using our PDP mix in a 1% mixture (Supplementary Fig. 4b), we found consistent accurate quantification was lost below 5% (Supplementary Fig. 4c). Thus, while PDP-based chimerism analysis is unlikely to supplant PCR based STR analysis for solution phase samples, it can serve as a complementing technology with the significant advantage of allowing the simultaneous in situ assessment of spatial relationships of donor and recipient elements. Mixing studies HapMap individuals with computationally discovered deletion polymorphisms via SNP analysis were selected based on gender and predicted copy number discordance at the autosomal loci (Supplementary data of McCarroll et al.) 1. The cellular densities of specimens were enumerated separately using a hemacytometer, and then subsequently the specimens were mixed together at the following ratios: 0-100, , 1-99, 5-95, 10-90, 50-50, 90-10, 95-5, and 100-0%. Slides were then prepared for FISH analysis, and assessment of chimerism was completed by counting at least 50 cells per mixture. For data analysis, the extent of chimerism as quantified by traditional XY-FISH was plotted against assessment of copy number status using the polymorphic deletion probes with a linear regression least-squares fit. Sensitivity 2

3 analysis was performed using serial dilutions of HapMap Chr2 PDP and Chr8 PDP discordant cell lines and quantification of chimerism was performed by the PCR-based short tandem repeat (STR) method using the AmpFlSTR Profiler Plus PCR Amplification Kit (Applied Biosystems) and analyzed using Genotyper software. Array CGH Genomic DNA was extracted from cell lines using the Puregene DNA Purification Kit according to standard procedures. A pooled normal male DNA (Promega) was used as reference, independent of the patient s gender. Array CGH was performed using Agilent Technologies 224k oligonucleotide arrays according to the recommended protocol (Agilent Technologies). Briefly, one microgram of DNA, and male reference DNA, was digested at 37 ºC for two hours using AluI and RsaI. Digested DNA was labeled by random priming (BioPrime Array CGH Labeling Module; Invitrogen) using Cy3-dUTP or Cy5- dutp (GE Healthcare). After an incubation of two hours at 37 ºC, labeled DNAs were purified using the Millipore Microcon YM-30 purification kit according to the manufacturer s instructions. Labeled tumor and reference DNA were combined together with Human Cot-1 DNA (Invitrogen) using the Agilent Oligo acgh Hybridization Kit. Subsequently, the DNA was denatured for 3 minutes at 95 ºC, pre-hybridized for 30 minutes at 37 ºC and hybridized for 35 to 40 hours at 65 ºC. After hybridization, slides were washed with Agilent Oligo acgh Wash Buffer 1 and Buffer 2, at room temperature for five minutes and at 37 ºC for one minute 3

4 respectively. These two washes were followed by a third wash in stabilization and drying solution. Washed slides were scanned using the Agilent G2565 Microarray Scanner. Data was extracted from the microarray TIFF images (.tif) using Agilent s Feature Extraction Software v9.1, and analysis was performed using Agilent s CGH Analytics software. Copy number alterations were considered significant if the log 2 ratio were ±2 standard deviations from the mean intensity of the entire experiment 2. All of the potential deletion loci possessed log 2 ratios of 4.0 or less. Calculation of informative potential of polymorphic deletion probes Assuming Hardy-Weinberg equilibrium for the distribution of independent deletion loci and using the binomial theorem, the non-informative probability of FISH testing for two PDP loci is the summation of all probabilities for which the two individuals have identical genotypes, (i.e. p 2 vs. p 2, 2pq vs. 2pq, and q 2 vs. q 2, with corresponding probabilities of pairings of p 4, 4p 2 q 2, and q 4, respectively). Schematically, this relationship can be considered using a 9 x 9 matrix showing the possible two PDP genotype combinations of two individuals of a chimeric pair, each shown along the orthogonal axes with deletion genotype frequencies for each of two PDP loci i and j following Hardy-Weinberg equilibrium: 4

5 Here, the shaded diagonal boxes represent the chimeric pairs that would be non-informative under PDP-FISH, as identical FISH results (+/+, +/, or / ) would be found for both the donor and recipient. In contrast, the non-shaded boxes (informative potential) represent pairings of two individuals that would result in informative PDP-FISH experiments. The informative potential of two PDPs, i and j, can therefore be calculated as: Informative potential = 1 [Non-informative probability] Informative potential = 1 [p 4 j (p 4 i +4p 2 i q 2 i +q 4 i )+ 4p 2 j q 2 j (p 4 i +4p 2 i q 2 i +q 4 i ) + q 4 j (p 4 i +4p 2 i q 2 i +q 4 i )] Using the relationship 1 = p + q, and assuming p i = p j, this relationship can be simplified to: Informative potential = 1- (6p 4-12p 3 +10p 2-4p+1) 2 5

6 For n probes, assuming p i = p j...= p n, Informative potential = 1- (6p 4-12p 3 +10p 2-4p+1) n This equation is used to generate Figures 4a and 4b. A similar analysis has been previously considered in part in the context of using bi-allellic SNPs for identity testing in bone marrow engraftment studies 3. Identical estimates for the maximal informative potential of three or four SNPs (~95% and ~98%, respectively) in distinguishing individuals of a chimeric pair were reported. 3 Estimates of null genotype frequencies For the Chr2p and Chr8p PDPs, the percent of individuals that are homozygously deleted can be estimated by analyzing the reported number of individuals with null SNP genotypes at these loci. 1 For the CEU HapMap population (European), null SNP genotypes were computationally identified in 14 out of 90 (Chr2p PDP) and 18 out of 90 (Chr8p PDP) individuals. Assuming Hardy-Weinberg equilibrium (p 2 + 2pq + q 2 = 1) for both deletion polymorphisms, one can calculate homozygous loss, null genotype, frequency at Chr2p PDP as 16% (p 2 ), heterozygosity as 48% (2pq), and homozygous no-loss as 37% (q 2 ). Similarly, for the Chr8p PDP locus, one can calculate homozygous loss, null genotype, frequency as 20% (p 2 ), heterozygosity as 49% (2pq), and homozygous no-loss as 31% (q 2 ). 6

7 SUPPLEMENTARY FIGURES Supplementary Fig. 1. General strategy for polymorphic deletion probe discovery. Null genotype deletion loci were searched for in the European (CEU) population using the Supplementary data of McCarroll et al. 1. Probes were selected on basis of size, reported frequency as a surrogate for population frequency, and location. Of note, the deletion loci not tested herein are not excluded as being potentially useful PDP targets. Using the UCSC genome browser, loci were mapped to select optimal fosmid clones with highest sensitivity and specificity. The presence of segmental duplications for the candidate loci is indicated in the right-most column. 7

8 a b c 8

9 Supplementary Fig. 2. Array CGH based PDP discovery and assessment of local genomic structure of deletion loci. Additional loci were identified based on our identification of recurrent deletion CNVs >25 kb using array CGH (a). Analysis by array CGH demonstrates the local genomic structure for the polymorphic deletion loci identified on chromosomes 4q (b) and 8p (c) showing loss at the identified loci of signal as compared to reference sample. Representation of the 2p locus was not included on the commercial array CGH platform used herein, and thus the corresponding local structure on 2p could not be explored by this method. 9

10 y = x R 2 = FISH by PDPs FISH by X,Y Supplementary Fig. 3. PDPs reliably detect extent of chimerism in idealized mixing studies. Correlation of chimerism as quantified by PDP-FISH versus traditional sex chromosome-based FISH of gender mismatched HapMap individuals (male NA10838 is Chr2p PDP (+/+) and Chr8p PDP (+/ ) and female NA10859 is Chr2p PDP ( / ) and Chr8p PDP (+/+)) with a linear least-square regression fit. At least 50 cells were counted at each mixture percentage. Filled and open circles denote chromosome 2 and 8 PDP results respectively. The X- axis denotes the percentage of cells composed of male individual NA10838 as detected by XY-FISH. The Y-axis denotes the percentage of cells composed of male individual NA10838 as detected by Chr2 and Chr8 PDP-FISH. 10

11 Supplementary Fig. 4. Sensitivity analysis of PDP FISH as compared to standard PCR-based short-tandem repeat analysis. Idealized mixtures of HapMap gender mismatched and PDP discordant cell lines (male NA10838 Chr2p PDP (+/+), Chr8p PDP (+/ )) and female NA10859 (Chr2p PDP ( / ), Chr8p PDP (+/+)) were assessed for chimerism using standard PCR based short-tandem repeat (STR) analysis. (a) The bottom tracing shows detection of the pure female DNA with absence of peak for the Y chromosome, whereas the top tracing is of pure male DNA with X and Y chromosome peaks. The middle tracing shows analysis of DNA from a one percent mixture of male cells diluted into female cells. Allele quantitation is shown by the peak height below each tracing. (b) Representative image showing that PDP-FISH based analysis is able to accurately detect male NA10838 in a 5% percent cell mixture with female NA10859 (green signal is Chr2p PDP, the red is the Chr8p PDP, and yellow is a Y chromosome probe used as an internal control). Scale bar is 10 microns. (c) Observed versus expected chimerism percentages using PDP-FISH. At least 50 cells were counted at each mixture percentage. 11

12 SUPPLEMENTARY TABLES Supplementary Table 1. Copy number loci and HapMap individuals examined. Copy number loci deletions as predicted by computational SNP analysis (from McCarroll et al. 1 ) and by array CGH analysis at selected loci (columns) were examined using the selected HapMap individuals (rows). Coordinates are from the hg16 (build 34) assembly of the Human Genome Project. Shaded columns represent deletion loci verified experimentally as true deletions in this study. For two of these columns (chromosomes 2p and 8p), HapMap individuals with predicted deletions are indicated and were verified. The remaining individuals in these two columns were found to have either no deletion or heterozygous deletions (these genotypes could not be differentiated by SNP analysis). 12

13 Supplementary Table 2. United Network for Organ Sharing data for the past 10 years on the gender of donors and recipients. Considerable gender skewing in donor-recipient pairs is seen for heart, lung, and heart-lung transplants, in which gender-matched states frequently occur, thus precluding the use of traditional XY-FISH for in situ chimerism assessment. In particular, malerecipient-and-female-donor transplant states which are most ideal for tracking the Y-chromosome in a female genetic background are of considerably lower frequency (~18%, 12%, and 12% respectively). PDP-FISH should substantially increase the number of chimeric transplant states that may be accessed, and may be more useful than XY-FISH for solid-organ transplantation (54% versus 46%). This data was provided by the Organ Procurement and Transplantation Network, ( based on data as of October 3, 13

14 2007. By report, this data analysis was supported in part by Health Resources and Services Administration contract C. The content is the responsibility of the authors (Wu et al.) alone and does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. 14

15 SUPPLEMENTARY REFERENCES 1. McCarroll, S.A. et al. Common deletion polymorphisms in the human genome. Nat Genet 38, (2006). 2. Iafrate, A.J. et al. Detection of large-scale variation in the human genome. Nat Genet 36, (2004). 3. Oliver, D.H., Thompson, R.E., Griffin, C.A. & Eshleman, J.R. Use of single nucleotide polymorphisms (SNP) and real-time polymerase chain reaction for bone marrow engraftment analysis. J Mol Diagn 2, (2000). 15