Developmental validation of a novel all-in one assay of X chromosomal multi-insertion/deletion loci for forensic genetics

Multi-insertion/deletion polymorphisms (Multi-InDels), as the novel genetic markers, show great potential in forensic research. Whereas, forensic researchers mainly focus on the multi-InDels on the autosomes, which can provide relatively limited information in some complex paternity cases. In this study, a novel X chromosomal multi-InDel multiplex amplification system was designed, containing 22 multi-InDels and one STR locus on the X chromosome. In order to verify the applicability and effectiveness of the novel system, the sensitivity, species specificity, repeatability, stability, and precision of the panel were verified. The results showed that the novel developed multiplex amplification system could still obtain complete allele profile at 62.5pg of template DNA and showed good tolerance to six common inhibitors. Meanwhile, the analyses of case samples showed that the system performed well on biological samples commonly found in forensic cases. The combined power of discrimination for males and females in the Guizhou Han population was greater than 0.999999999. Besides, the combined mean exclusion chance of these 23 loci exceeded 0.9999 in the trios and the duos. In conclusion, the multiplex amplification system is sensitive, precision, reproducible, and informative, which could be used as a high-efficient tool for complex kinship identification and personal identification.