Development and validation of a custom panel including 114 InDels using massively parallel sequencing for forensic application

Abstract

Insertion/deletion polymorphisms (InDels) have particular characteristics, such as a relatively low mutation rate, small amplicon size, and no stutter artifacts when genotyped via the capillary electrophoresis platform. It would be an important complementary tool for individual identification and certain kinship analyses. At present, massively parallel sequencing (MPS) has shown excellent application value in forensic studies. Therefore, in this study, we developed a custom MPS InDel panel that contains 114 InDels [77 autosomal InDels (A‐InDels), 32 X‐chromosomal InDels (X‐InDels), and 5 Y‐chromosomal InDels) based on previous studies. To assess this panel's performance, several validation experiments were performed, including sensitivity, inhibitor, degraded DNA testing, species specificity, concordance, repeatability, case‐type samples, and population studies. The results showed that the lowest DNA input was 0.25 ng. All genotypes were obtained in the presence of 80 ng/µL humic acid, 2000 µmol/L calcium, 3000 µmol/L EDTA and indigo. In degraded DNA testing, 90% of loci could be detected for 16‐day‐old formalin‐fixed hearts. In addition, this panel has good species specificity. The values of combined power of discrimination and the combined power of exclusion for 77 A‐InDels were 1–3.9951 × 10−32 and 1–4.2956 × 10−7, respectively. The combined mean exclusion chance for 32 X‐InDels was 0.99999 in trios and 0.99904 in duos. The validation results indicate that this newly developed MPS multiplex system is a robust tool for forensic applications.

Improving DNA mixtures analysis using compound markers composed of InDels and SNPs screened from the whole genome with next‐generation sequencing

“…However, this concept is not widely accepted. The main challenge comes from the nature of InDel, especially the variable and complex alleles such as duplications and homopolymers, which often make sequence alignment difficult [47]. In this study, we intentionally avoided the publicly available InDels with complex alleles, and selected short InDels that are easy to detect and analyze to integrate with one or more adjacent SNPs as compound markers for analysis.…”