Application of autosomal INDELs as a forensic tool in Qatar

Bashir, Majid (2016) Application of autosomal INDELs as a forensic tool in Qatar. Doctoral thesis, University of Central Lancashire.

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Short tandem Repeats (STRs) are the most commonly genetic markers used in forensic human identification. However, in some cases they are not able to yield complete profiles because of DNA degradation and/or inhibition. The STR profiling of the degraded/inhibited DNA samples can result in allelic drop-outs and even no profile at all. Alternatively, single nucleotide polymorphisms (SNPs) can be used to address the issues of DNA degradation and inhibition due to their smaller amplicons. But their use in regular forensic case work is limited due to additional steps (sequencing based) and time consuming process. To bridge the gap between STRs and SNPs by combining their characteristics, another type of genetic marker, insertion deletion polymorphisms (INDELs), offer an effective way to analyze challenged DNA sample (degraded and inhibited ones). INDELs have short amplicons, low mutation rates, no stutter peaks and are analyzed using the same equipment and protocols as STR polymorphisms.
In this study, the forensic efficiency of 30 autosomal INDELs contained within the Qiagen™ Investigator™ DIPplex kit were tested by using 500 samples from individuals belonging to five different nationalities (Qatari, Pakistani, Sudanese, Tunisian and Yemeni) based in Qatar. Population indices and forensic parameters were calculated. The results showed no significant deviation from Hardy-Weinberg Equilibrium and no evidence of linkage disequilibrium for all of 30 markers was found after applying Bonferroni’s correction (P < 0.00166). The Combined Power of Discrimination (CPD) for the 30 INDEL loci was 0.9999999 for all of five populations which shows that these 30 markers are very efficient and suitable for forensic casework. The Combined Probability of Match (CPM) was calculated in the order of 10-13 which is a satisfactory value for forensic purposes. In addition to assess forensic efficiency of 30 autosomal INDELs, an effort was also made to derive ancestry information by using different statistical systems (Arlequin, Snipper and STRUCTURE). The results indicated that 30 INDELs contained in Qiagen™ Investigator® DIPplex kit, although useful for forensic identification, poorly differentiate five population groups of Qatar. The reason of failure in differentiating the populations, lies in the selection of INDEL markers, which were chosen for identification purpose (i.e. they have similar allele frequencies in different populations) rather than deriving the ancestry information (i.e.
ancestry informative markers are chosen on the basis that their allele frequencies need to be different for different populations).
In order to recover genetic information lost during DNA degradation, the concept of reduced sized PCR products (mini amplicons) has been developed. MiniFiler® kit (Applied Biosystems™) containing 8 mini-STRs (developed by re-designing the primers of 8 STRs contained in Identifiler Plus® kit) provide a possible solution of DNA degradation. By using similar mini-plex approach, a multiplex PCR assay for INDELs (named as mini-INDELs) has been developed in this research. A total of 14 autosomal INDEL markers were selected and short amplicons were designed for them keeping in view that they could perform efficiently on degraded samples. The multiplex of 14 INDELs was designed and optimized successfully in a single tube reaction. All the markers were amplified adequately with good peak balance and expected amplicon sizes. The sensitivity of mini-INDELs was found upto 0.03125 ng of genomic DNA with complete and balanced profile. The concordance between mini-INDELs kit and Qiagen™ Investigator™ DIPplex kit (for the common loci) was observed in 99.7% INDEL alleles. The efficiency of mini-INDELs PCR assay was also evaluated on a set of artificially prepared degraded and inhibited DNA samples.
It is concluded that INDELs markers in general and mini-INDELs in specific, can be used as a useful tool in forensic case work and can also be employed in conjunction with STR typing as a complementary tool especially in those cases where low level of DNA and DNA degradation are suspected.

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