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Objective: In a paternity case, the D12S391 locus was reported as a mismatch. To confirm the existence of mutations and mutations come from father or mother. Methods: STR and next-generation sequencing technology were used to validate the sequence. Results: NGS showed the loss of one adenine between the 19.3 allele of the child and allele 20 of the mother. Conclusion: The NGS can be applied in the paternity to validate the mutation.

Half-sibling (HS) kinship testing is more difficult and complicated.This article describes a case where we sought to establish if three children with different mothers (one boy and two girls), shared a common father. The three children and their mother,as well as the suspected father's elder brother took part in the identification. To investigate the application of multiple genetic markers in HS kinship testing,autosomal STR, Y-STR, and X-STR was performed on blood samples from the individuals of interest C1 (female), M1 (mother of C1), C2 (male), M2 (mother of C2), C3 (female), M3 (mother of C3), and UC (elder brother of the suspected biological father). The 24 Y-STR haplotypes of C2 and UC were identical, confirming that they come from the same paternal line.Among the 27 X-STRs tested in C1 and C3, 18 of them had different paternal alleles, so C1 and C3 were excluded as half-sibling kinship.39 autosomal STR were calculated by ITO method,the HSI (or AI) of the pairs C1–C3, C2–C3, and C3–UC are 1, which may supports HS kinship between C1 and C2 and avuncular kinship between C1 and UC, and between C2 and UC.15 autosomal STR were calculated by discriminant function, DHS3 > DR3 between C1 and C2, and thus they could be HS. DHS3