Assigning individuals to ethnic groups based on 13 STR loci X. Fosella 1, F. Marroni 1, S. Manzoni 2, A. Verzeletti 2, F. De Ferrari 2, N. Cerri 2, S. Presciuttini 1 1 Center of Statistical Genetics, University of Pisa, Pisa, Italy 2 Institute of Legal Medicine, University of Brescia, Brescia, Italy

Introduction  Inferring individual’s ethnic origin by means of short tandem repeat (STR) profiles has recently received considerable attention in several fields of applied genetics  Gene frequency variation among human populations has been extensively documented, and it has been suggested that the differences in allele proportions between ethnic groups could form the basis of an inferential system  We selected several groups of ethnically defined immigrants in a province of Northern Italy and assessed the power of 13 STR of tracing back their origin

Backgrounds of this study  Brescia ranks among the highest towns in Italy for the proportion of censused immigrants from non-EU countries (about 10% of the local resident population)  Most blood crimes in this area happens within ethnically defined groups  A test that could assign a biological stain to a subject of a particular population would be highly welcomed

Population samples Unrelated individuals from the historical series of the Institute of Legal Medicine of the University of Brescia, including a large number of immigrants from a variety of countries, were selected for the present analysis Unrelated individuals from the historical series of the Institute of Legal Medicine of the University of Brescia, including a large number of immigrants from a variety of countries, were selected for the present analysis In addition, blood samples from subjects of known ethnicity were collected from the local hospital, following IRB approval In addition, blood samples from subjects of known ethnicity were collected from the local hospital, following IRB approval The composition of the final sample is shown below The composition of the final sample is shown below Subjects were typed with the Profiler Plus TM and SGM Plus TM kits (totaling 13 loci), following standard protocols Subjects were typed with the Profiler Plus TM and SGM Plus TM kits (totaling 13 loci), following standard protocols

Population assignment test (1) Theoretically, assigning individuals to a given population based on a multilocus genotype is surprisingly easy.

Population assignment test (2) A computational trouble (a division by zero) arises when a particular allele is absent from a population sample; in this case, an arbitrary frequency must be given to that allele in that population.

A popular program in genetic data analysis The software Arlequin ( arlequin/) includes a routine for computing the likelihood of each multilocus genotype of a defined population sample in each of a number of specified samples. We first applied Arlequin’s calculation to all our data; however, the assumption made by the program about the “missing” alleles is not clear. Therefore, we analyzed in more detail the comparison Blacks/Italians using an explicit and conservative assumption.

Arlequin results These charts show the individual log- likelihoods of four samples of immigrants plotted against the log- likelihood of Italians being falsely attributed to each of these samples

A closer look at the comparison Blacks vs Italians The analysis was repeated for these two samples with our algorithm, using the allele frequencies estimated from all available data (about 100 subjects typed for each locus in both samples), and assigning a value of 1/200 (=0.005) to the frequency of the missing alleles

Simulated samples In order to estimate the statistical power of the assignment test with higher accuracy, we simulated 10,000 individuals for both the Italian and the Black samples

Statistical power of discriminating between the two simulated samples (Blacks vs Italians)

Conclusions (1) The 13 STR loci included in two commercial kits provided a limited but significant power to infer the ethnicity of immigrant groups. The 13 STR loci included in two commercial kits provided a limited but significant power to infer the ethnicity of immigrant groups. Not surprisingly, the highest level of discrimination was achieved by contrasting Blacks with resident Whites. Not surprisingly, the highest level of discrimination was achieved by contrasting Blacks with resident Whites. When two alternative hypotheses about the ethnic origin of a sample can be formulated with confidence, a population assignment test can already be applied to real cases When two alternative hypotheses about the ethnic origin of a sample can be formulated with confidence, a population assignment test can already be applied to real cases

Conclusions (2) The present work suggests that a sequential test can be highly efficient in assigning individuals to their ethnic group; given a fixed pre-defined significance level, investigators can start typing a small number of markers, and typing additional marker sets only for the subjects that failed to be classified at the previous step. The present work suggests that a sequential test can be highly efficient in assigning individuals to their ethnic group; given a fixed pre-defined significance level, investigators can start typing a small number of markers, and typing additional marker sets only for the subjects that failed to be classified at the previous step.