Construction of A Novel 5‐Dye Fluorescent Multiplex System With 30 Y‐STRs for Patrilineal Relationship Prediction

Nowadays, Y chromosome short tandem repeats (Y-STRs) are widely used in forensic medicine practice, which has great significance for ancestry tracing, male lineage evolution, and male paternal relatives. Rapidly mutating Y-STRs (RM Y-STRs) have been shown to have greater potential to distinguish males from males in the patrilineal line. Therefore, a novel 5-dye fluorescent multiplex system with 30 Y-STRs was developed and optimized to screen out more RM Y-STRs and fasting mutating Y-STRs (FM Y-STRs). New primers were designed, and composite system construction was carried out. A series of experiments were conducted following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), including polymerase chain reaction (PCR) amplification conditions, sensitivity, stability, species specificity, mixture and degraded sample studies, mutation analysis, and population studies. The results suggested that changing PCR amplification conditions in a reasonable range hardly affected the genotyping. The system showed excellent sensitivity and stability in sensitivity and stability studies. Even with UV-C exposure for up to 96 h, the system performed well in male blood samples and semen-vaginal secretion mixtures. Mutation analysis was performed on 582 father-son pairs, and 4 RM Y-STRs and 7 FM Y-STRs were identified, with mutation rates ranging from 1.72 × 10^-3 to 20.62 × 10^-3. Furthermore, on the basis of mutation rate analysis, eight machine learning methods were used to construct and compare patrilineal relationship prediction models, inferring the relationship by predicting the number of meiosis. Overall, the multiplex system displays favorable performance and has a greater potential for application in forensic science practice.