2450 Nucleic Acids Research, 2008, Vol. 36, No. 7 Figure 2. Schema of SNP panel identiﬁcation assay (SPIA) applicability and use modality. One typical scenario would be to check out of the identity of a cell lines (SAMPLE 1) with respect to already ﬁngerprinted cell lines, the data of which are stored in the BARCODING REFERENCE database. Another scenario would be to determine the identity of two cell lines, for example derived from the same patients, one from benign tissue cells and one from cancer tissue cells (SAMPLE 1 and SAMPLE 2). First, the DNA is extracted from the cell line(s) and is experimentally genotyped along the set of speciﬁed loci included in the SNP panel. Sequenom mass spectrometric technology, allele-speciﬁc PCR or other techniques can be implemented. Afterwards, the allele call output sets (DNA ﬁngerprints) are compared. Comparison is performed pair-wise. Comparison with all the ﬁngerprints stored in the reference database can be performed on demand. The SPIA test provides the genotype distance between the two samples and detailed information on the allele mismatches. It also provides the user with a probability measure of the test output, scoring the tested pair as ‘similar’ or ‘diﬀerent’. If the test result is uncertain, the assay can be repeated using an additional SNP panel. such as matching human tumor samples with their non-neoplastic normal tissue. The resulting assay, termed the SNP panel identiﬁcation assay (SPIA), allows investigators to accurately identify known cell lines and tumors from the genotype of extracted DNA. SNP panel selection To deﬁne the optimal SNP panel, we reasoned that the ideal SNPs should collectively maximize the probability of obtaining distinct genotype calls on diﬀerent samples, i.e. exhibiting the greatest heterogeneity across samples. SNPs with two alleles (A and B) give rise to possible genotype calls AA, BB and AB. Hypothetically, genotype frequencies equal to one-third each should maximize the probability of obtaining distinct genotyping calls on diﬀerent samples, with 9 SNPs being suﬃcient to distinguish 20 000 samples (see Supplementary Material ‘How many SNPs do we need to have a robust ﬁngerprint’). However, in a given population, SNPs under neutral selection are in Hardy–Weinberg equilibrium, in which allele frequencies ﬁt the equation P2 + 2PQ + Q2 = 1. Since the vast majority of SNPs in the human genome approximate Hardy–Weinberg equilibrium, the optimal genotype probabilities are PAA = 0.25, PBB = 0.25, PAB = 0.5 for genotype calls AA, BB and AB, respectively. The SNP panel will have the most power to distinguish individuals when the selected SNPs are independent of one another. This can be accomplished by selecting SNPs from diﬀerent chromosomes and/or SNPs from the same chromosome that are not in linkage disequilibrium. If all human cell lines were genotyped, one could rank the SNPs that best distinguish these cell lines from the entire set based on the call frequencies. Iterative analysis