Sample collection and DNA extraction
Two male (Chhotu and Mastan) and one female (Buri) Chimpanzee hair samples were received from the Alipore Zoological Garden, Kolkata. These Chimpanzees were juvenile and were confiscated by the officials of the Wildlife Crime Control Bureau (Eastern Unit). Genomic DNA extraction was carried out from the hair follicles using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Germany), following the manufacturer's protocol.
PCR amplification and Genotyping
Three most widely used 6 dye multiplex Human STR systems i.e. GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C System were used for the genotyping of the samples. The GlobalFiler™ PCR Amplification Kit includes 21 autosomal STR markers i.e. D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, D2S1338 with Amelogenin (AMEL) as a gender determining marker, DYS391 as Y STR marker and one Y-indel polymorphic marker. The Investigator 24plex QS Kit includes 22 CODIS recommended STR markers TH01, D3S1358, vWA, D21S11, TPOX, DYS391, D1S1656, D12S391, SE33, D10S1248, D22S1045, D19S433, D8S1179, D2S1338, D2S441, D18S51, FGA, D16S539, CSF1PO, D13S317, D5S818, D7S820 including two quality control sensors QS1 and QS2 and a gender determination marker Amelogenin. The PowerPlex® Fusion 6C System following the recommendations of both CODIS and ESS consists of 18 expanded CODIS core loci i.e. CSF1PO, FGA, TH01, vWA, D1S1656, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11 and five highly discriminative loci i.e. Penta D, Penta E, D22S1045, TPOX, and SE33. It also includes two gender determining markers that are Amelogenin and DYS391 and two rapidly mutating Y-STR markers i.e. DYS570 and DYS576. For each marker system, separate PCR reactions were carried out in a total volume of 25 μl with both positive and negative controls on the GeneAmp PCR system 9700 thermocycler (Applied Biosystems, USA) following the manufacturer's protocols. Fragment analyses of the amplified products were carried on ABI 3130 Genetic analyzer (Applied Biosystems, USA).
Genotype calling was performed using GeneMapper ID v.3.2 and the scoring and rearrangement of allelic data were performed in Microsoft Excel. Genetic diversity indices such as Observed heterozygosity (Ho), Expected heterozygosity (He), Unbiased heterozygosity (uHe), Observed number of alleles (Na), Expected number of alleles (Ne), and Fixation Index (F) were obtained using GENEALEX v.6.5 . Since the individuals were confiscated animals, therefore genealogical analysis to establish the extent of the relationship was critical. Hence, the likelihood of relatedness between the individuals was estimated using ML-Relate .