0005631 (3/5328), in a paternity index of 1776 (1/0.0005631) and in a probability of paternity of 99.9437%.

The DYS385 locus was excluded from the quantitative analysis in the cases with dropout (3, 17, and 18) and it did not change the number of matches in the database. There was total match between the newborn Y-STR haplotype and the Y-STR loci detected in the maternal plasma in all 20 cases with male fetuses (Table S1). Previous studies have successfully amplified Y-STR from maternal plasma by using commercial kits, howsoever, the haplotypes Bleomycin retrieved was not consistently extensive enough with 6–16 Y-STRs, 12 on median [25] or 5–12 Y-STRs, 8 on median [26] to be high discriminatory. Consequently, they would have higher frequency compared to haplotypes found in the present study, which are associated with lower paternity index and probability of paternity. The consistent obtainment of such extensive haplotypes was possible due to different reasons: (a) there were substantial

overlap between the loci included in the multiplex systems; (b) the high amplification cycle number compared to previous studies [25] and [26]; (c) the 3500 Genetic Analyzer had several significant changes from the previous 31xx generation instruments [27]; and (d) the high input of maternal plasma (1 mL) used for DNA extraction. The use of high amplification cycle number is a standard procedure in the non-invasive pre-natal AZD5363 diagnostic. Previous studies in the field have described PCR amplification step with 60–50 PCR cycles [1], [28], [29] and [30]. Nonetheless, this procedure together with the capillary electrophoresis analysis Methane monooxygenase is prone to artifacts like nonspecific amplification and color pull-up that results in drop in (see Figs. S1 and S2). Therefore, great care should be taken in the profiles interpretation (see DYS 549 locus of the Powerplex Y23 profile at Fig. S1, it was excluded from the analysis due to the allele 12 drop in, despite the allele 13

match the alleged father profile). Furthermore, the high amplification cycles number is also prone to PCR contamination; the known procedures to avoid amplicon carryover should be applied strictly. The use of only mini Y-STR, which allows the use of less amplification cycle number should eliminate this problem. Today, in our complex society, there are many situations where it would be desirable to perform the non-invasively prenatal paternity testing by the analysis of the circulating cell-free fetal DNA (e.g. ambiguous paternity in case of women with more than one sexual partner who are unsure of the actual father) [8], [31] and [32]. The fetal male lineage determination by analysis of Y-chromosome STR haplotype in maternal plasma described in this study can be use as an alternative for this purpose.