In order to know the gender of an individual using STR analysis, there is a locus on sex chromosomes called amelogenin having a region containing six base pairs longer in males than females. PCR amplification of female samples will show one band on agarose gel for amelogenin since they have two X chromosomes, while males result will indicate two amelogenin bands, one that is six base pairs longer than the other, as they have one X and one Y chromosomes (Figure 2) .
Due to the nature of degradation that crime scene samples have because of the exposure to different environmental conditions , STR technique may not have the ability to produce a complete profile. The length of target amplicons of STR range from 100 400 base pairs (bp), while it can be 200 bp in highly degraded samples . In addition to the large amplicon size and the high possibility of yielding a partial profile for highly degraded samples, STRs have a relatively high mutation rate, which may affect the analysis of complex kinship cases (Figure 3) .
These factors contribute to the potential limitations of STRs in forensic applications. Therefore, it is necessary to use alternative methods such as miniSTRs, single nucleotide polymorphisms (SNPs), and insertion/deletion polymorphisms (indels) .
1.3.2 Additional Genetic Markers in Forensic Science
Autosomal STR markers have become the most commonly used markers throughout the forensic laboratories in the world. However, there are some cases that require cross-examination with alternative markers. Genetic variations in the form of DNA polymorphisms, including single nucleotide polymorphism (SNP) and insertion-deletion polymorphism (indel), are useful due to their ability to target shorter template sequences (smaller amplicons) . They have become complementary markers that can be considered for human identification purposes and forensic applications.
126.96.36.199 Single Nucleotide Polymorphisms (SNPs)
SNPs are single base pair variation at a particular location on an average of every few hundred base pairs in the human genome. So, they are typed as a sequence variation as illustrated below .
SNP markers require a small size of the target DNA to be recovered compared to STRs. This major advantage makes them valuable for the analysis of degraded DNA samples and disaster victim identification .
On the other hand, it is not recommended to replace the SNPs with STRs because there are many disadvantages associated with the application of SNP loci. First of all, the discrimination power of SNP markers is very low compared to STR loci. It is necessary to include 50 SNPs in the assay in order to reach match probabilities of 10 STR markers . In addition, It is difficult to use SNP markers for the mixture analysis because it is not easy to distinguish between mixtures and homozygotes, particularly for minor contributors, and which may show possible allelic dropout .
Although the use of SNP technique has been of high value in certain applications, such as analysis of highly degraded samples , it was shown that it is not practical in large scale of forensic analysis because they require complicated procedures with many steps, assays and expensive instruments , . Therefore, SNPs technique is hard and time consuming , as it includes additional steps compared to STRs (Figure 4). Yet, SNP markers have proved to be helpful in other applications, including the phenotypic features determination, which may help the forensic field, such as eye color and hair color by indicating the variance in the melanocortin 1 receptor (MC1R) gene .
188.8.131.52 Insertion-deletion Polymorphisms (Indels)
Insertion or deletion of bases in the genome is a type of genetic variation termed as indel. It is the result of either insertion or deletion of a DNA sequence ranging from one nucleotide to hundreds of nucleotides (Figure 5) , , . It is also known as a form of bi-allelic (or di-allelic) polymorphisms in which the two alleles can be typed as short and long .
STR loci can be classified as multiallelic indels since the allelic difference is classically the insertions or deletions of a tandem repeat element. Most bi-allelic indels show allele-length variations of only a small number of bases . Over 2000 bi-allelic indels in the human genome have been recently indicated by James Weber and colleagues at the Marshfield Medical Research Foundation. They described that 71% of these indels have 2-, 3-, or 4-nucleotide length differences and only 4% have more than a 16- nucleotide length difference .
Indels, which are bi-allelic insertion/deletion polymorphisms also known as DIPs, have many advantages , . They have low mutation rate compared to STRs which makes them applicable for complex kinship analysis , . They represent approximately 15.6% of all polymorphisms found in human genome, proposing that there is a minimum of 1.56 million indels within human genome , , so they are widely spread throughout the human genome . They have short amplicon size, which is useful for degraded or ancient samples with low template DNA . Moreover, they have the characteristic of the absence of stutter peaks and microvariant products which facilitates the interpretation of mixture samples , . They can also be used as ancestry informative markers (AIMs) to study substructure in mixed populations , . Additionally, they can be analyzed easily using simple protocols and automations already available and currently used in STR typing in forensic laboratories (Figure 4) as well as have the property of high multiplexing capacity , . Overall, indels combine the characteristics of STRs and SNPs which make them a strong supplemental tool for human identity testing .
Indel allelic frequencies have been determined in different populations including African, European, Japanese, and Native American. A multiplex assay of 38 bi-allelic autosomal indel markers detected by standard protocols of capillary electrophoresis has been discovered by Rui Pereira and colleagues in Portugal and Spain, which helped to obtain complete profiles from a DNA template with approximately 300pg concentration . In addition, all indel markers were polymorphic in 306 individuals from Angola, Mozambique, Portugal, Macau, and Taiwan and gave random match probabilities RMPs of 10-14, which is similar to about 13 STR markers , .
1.4 Discovery of Indels
1.4.1 Indels Characteristics and Classification
Indels were originally identified on chromosome 22 in the human genome. 13% of the data obtained from the re-sequencing of this chromosome for 31 humans showed genetic variations known as indel polymorphisms , .
There are many characteristics which make indels useful for forensic studies; about 1.4 million human indels have been recognized and classified in the human genome , , the availability of indels in human genome gives them the power of distinguishing one individual from another which supports human identity testing. Approximately there are 344,000 indels present in human genome i.e., around 1 indel per 7.2 kb .
The calculation of mutation rates between individuals are based on the frequency of mutation occurrence and their probabilities in an individual compared to their parents. Indels have low mutation rates, which make them powerful in relationship and identity testing . Both indels and SNPs have similar mutation rates in the range of 1 in 107 to 108 meiosis events , . However, STRs have 1 to 4 per thousand meiosis mutation rates , ,  (Figure 6). The lower the mutational rate is, the more accurate paternity and kinship analysis results will be , , . Therefore, paternity index results can be improved using lower mutation rate markers.
Indels have been classified into five classes such as: (1) insertion and deletion of single-base pairs, (2) monomeric base pair expansions (3) multibase pair expansions of 2-15 bp repeat units, (4) transposon insertions and (5) indels containing random DNA sequence between 2-9800 bp constitute 40% of human genome. More than 99% of indels are shorter than 100bp , .