Furthermore, Galunisertib order Yfiler and Powerplex Y23 overlap in 9 successfully detected loci (Table 2, 5th column) and, despite the redundancy between both systems, Yfiler was the major contributor for the detection of 7 Y-STR (Table 2, 2nd column), because the amplicon size for these loci were smaller in the former one compared with Powerplex Y23 (Table S2), e.g. the amplicon

size of DYS19 ranges from 167 bp to 218 bp in Yfiler and in Powerplex Y23 from 312 bp to 352 bp. However, we cannot exclude that this result is secondary to the fact that in 8 out of 20 cases two Yfiler reactions were performed. The Y-STR haplotype detected in maternal plasma completely matched the alleged father in 16 out of the 20 cases and 4 cases showed singles mismatches (Table 1). Fig. S4 showed a representative example of matching analysis between maternal plasma and alleged father Y-STR haplotypes. In short, the extensive haplotypes retrieved from the maternal plasma resulted in an overall concordance at Y-STR loci PF-01367338 in vitro level of 99.2%. In regard of the mismatches, they were: (a) The case 1 that showed a single exclusion/mutation pattern at DYS458 due to the loss of one repeat unit. The kinship analysis of the case 1 (trio), performed after the delivery by using the autosomal STR markers included in the NGM kit (life Technologies) and local allele frequency population data [22], confirmed the paternity (paternity index of 3,472,249,188.76

and probability of paternity of 99.999999971). The mutation was also confirmed after the delivery

by using the Powerplex Y23 (Fig. S5). Indeed, the probability of find at least one mutation between two Y-STR haplotypes one generation apart, if 22 and 26 loci were genotyped, is relatively high, 6% and 7%, respectively [23]. The number of Y-STR locus surveyed in the YHRD in each case ranged from 13 to 16, and the median was 16 (Table S3). Quantitatively, a paternity index and a probability of paternity were attributed to each case. These estimations were based on the fetus haplotype frequency retrieved from Brazilian national database found in YHRD. In 16 out of the 20 cases, the fetal haplotype did not match any of the 5328 Brazilian haplotypes available at the YHRD, that resulted in a haplotype ADAMTS5 frequency of 0.0001877 (1/5328), in a paternity index of 5328 (1/0.0001877), exactly the database sizes, and in a probability of paternity of 99.9812%. In 1 out of the 20 cases, the fetal haplotype did not match to any Brazilian haplotypes available at the YHRD, but has a mutation in DYS 458 locus. In this case, the haplotype frequency was 0.0001877 (1/5328), paternity index was calculated by the specific formula described in methods [(0.5 × 0.00836)/0.0001877 = 22.271], which included a penalization that accounted the mutation rate of the DYS458 locus (0.00836) [24], and this paternity index resulted in a probability of paternity of 95.7028%.