Muhammad Israr

Y-chromosomal STR analysis in the Pashtun population of Southern Afghanistan

Afghanistan is a landlocked country in the heart of Asia and since the dawn of humankind Afghanistan has faced centuries of turmoil, strife, conflict, warfare, distress, social unrest, difficult climate, harsh terrain and due to its unique geostrategic position in Eurasia which has historically attracted commerce and conflict. It is an important stop along the Silk Road, connecting the far eastern civilizations to the western world. A 5000-year history of constant invasion. Afghanistan has been repeatedly invaded and conquered by rulers and super powers, neighboring interference in this conflict-tattered land for centuries yet rarely leading to the conquest of this rugged and challenging terrain nation. Afghans are not only shepherds, farmers and nomads but also intense fighters and fierce warriors. Currently very limited genetic studies have been performed in Afghan populations. 17 Y chromosomal short tandem repeats (Y-STRs) were analyzed in 125 unrelated Pashtun (in hindi: Pathan) males residing in the Kandahar region of Southern Afghanistan. A total of 92 unique haplotypes were observed. The predominant haplotype reached a frequency of 9.6%. The haplotype diversity was 0.987 and the discrimination capacity 73.6%. Analysis of molecular variance (AMOVA) reveals a considerable regional stratification within the country as well as between different Pashtun (Pathan) groups from Afghanistan, Pakistan and India.

Y-STR haplotype diversity in Punjabi population of Pakistan

We studied the genetic diversity of 17 Y-chromosomal STR loci (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and Y-GATA H4) in a sample of 300 genetically unrelated males from Punjabi population of Pakistan. DNA was extracted from blood stains collected on blotting paper (Whatman, Kent, UK) using Phenol chloroform extraction method [1] and quantified using QuantifilerHuman DNA Quantification Kit using ABI 7500 Real-time PCR System. Y-chromosomal STR loci were amplified using AmpFLSTR Yfiler PCR kit with GeneAmp 9700 PCR System (Life Technologies Inc.) according to the manufacturer’s instructions. Amplified products were analyzed by using the ABI PRISM 3100 Genetic Analyzer according to the manufacturer’s recommended protocol. ABI GeneScan 500-LIZ was used as internal size standard along with ABI Hi-Di Formamide. The data were analyzed using ABI GeneMapper ID v3.2. Alleles were designated according to the latest recommendations of the International Society for Forensic Genetics [2]. Haplotype and Allele Frequencies were estimated by simple counting using a spreadsheet program. Discrimination Capacity (DC) of the haplotypes was calculated as DC = H/n, where H is the number of different haplotypes and n is the number of total samples. Estimation of haplotype and gene diversities together with intraand inter-population indices was computed using ARLEQUIN v3.5 [3]. Analysis of molecular variance (AMOVA) was calculated using the AMOVA tool available at YHRD website (www.yhrd.org/Analyse). Total 274 different haplotypes were observed, in which 254 were unique while 20 were observed more than once (Supplementary Table S1). The discrimination capacity was 91.33% while unique haplotypes accounted for 92.7%, showing that these loci are useful for paternity testing and personal identification in the Punjabi population. Locus DYS385 showed the highest diversity (0.9171) while locus DYS391 showed the lowest diversity (0.4137). Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.12.004. Punjab (Persian; five waters), is the largest province of Pakistan by population accounting for 56.2% of the country’s population of 180 million (www.census.gov.pk). It is believed that urban civilization existed in the region before the Aryans invasion in around 2500 BC. Occupying a strategically key location, the region has remained part of many empires and civilizations during the course of history. It remained an important hub of Mughal Empire followed by a brief rule by the Sikhs before it fully came under the British Rule and later became part of Pakistan [4]. We compared our data to that of 10 other populations: Afghanistan [Pathan] [5],

EMPOP-quality mtDNA control region sequences from Kashmiri of Azad Jammu & Kashmir, Pakistan.

The mitochondrial DNA (mtDNA) control region (nucleotide position 16024-576) sequences were generated through Sanger sequencing method for 317 self-identified Kashmiris from all districts of Azad Jammu & Kashmir Pakistan. The population sample set showed a total of 251 haplotypes, with a relatively high haplotype diversity (0.9977) and a low random match probability (0.54%). The containing matrilineal lineages belonging to three different phylogeographic origins of Western Eurasian (48.9%), South Asian (47.0%) and East Asian (4.1%). The present study was compared to previous data from Pakistan and other worldwide populations (Central Asia, Western Asia, and East & Southeast Asia). The dataset is made available through EMPOP under accession number EMP00679 and will serve as an mtDNA reference database in forensic casework in Pakistan.

Forensic SNP Genotyping with SNaPshot: Development of a Novel In-house SBE Multiplex SNP Assay,

This study introduces a newly developed in‐house SNaPshot single‐base extension (SBE) multiplex assay for forensic single nucleotide polymorphism (SNP) genotyping of fresh and degraded samples. The assay was validated with fresh blood samples from four different populations. In addition, altogether 24 samples from skeletal remains were analyzed with the multiplex. Full SNP profiles could be obtained from 14 specimens, while ten remains showed partial SNP profiles. Minor allele frequencies (MAF) of bone samples and different populations were compared and used for association of skeletal remains with a certain population. The results reveal that the SNPs of the bone samples are genetically close to the Pathan population. The findings show that the new multiplex system can be utilized for SNP genotyping of degraded and forensic relevant skeletal material, enabling to provide additional investigative leads in criminal cases.

A comprehensive Y-STR portrait of Yousafzai’s population

In the current study, 17 Y-Chromosomal short tandem repeats (Y-STRs) included in theAmpFlSTR Y-Filer amplification kit (Applied Biosystems, Foster City, USA) were investigated in 146 unrelated Yousafzai males residing in the Khyber Pakhtunkhwa Province of Pakistan. A total of 94 (89.52%) unique haplotypes were observed. Discrimination capacity was 71.92%. Haplotype diversity ranged from 0.354 (DYS456) to 0.663 (DYS458). Both Rst pairwise analysis and multidimensional scaling plot showed that the genetic structure of the Yousafzais is significantly different from neighbouring populations.

Comparative study of STR loci for typing old skeletal remains with modified protocols of AmpFlSTR Identifiler and AmpFlSTR MiniFiler STR Kits

This study highlights a comparative study of short tandem repeat (STR) loci with modified protocols of AmpFlSTR Identifiler and AmpFlSTR MiniFiler STR Kits for typing 27 old skeletal remains collected from 100–1000-year-old mass graves in Pakistan. DNA profiles were obtained from minute quantities of DNA (even from ≤ 10 pg/μL) with modified protocols of these kits, which is a significant achievement in this study. Consensus profiles were produced for each bone sample. A comparison was carried out between Identifiler and Minifiler successfully genotyped STR loci. Full concordance was perceived in 97.33% (146/150) of the compared STR loci, while discordant STR loci were 2.67% (4/150) of the total successfully genotyped STR loci due to either or both allele drop-out or drop-in. Finally, it was observed that the AmpFlSTR MiniFiler kit promoted the recovery of locus/alleles that failed to type with the AmpFlSTR Identifiler kit and more informative DNA profiles were obtained from old skeletal remains with the AmpFlSTR MiniFiler STR kit compared with the AmpFlSTR Identifiler STR kit.

Forensic DNA Typing of Old Skeletal Remains Using AmpFlSTR ® Identifiler ® PCR Amplification Kit

Background: In this study DNA typing of old skeletal remains was improved through AmpFlSTR®Identifiler®PCR amplification kit using different approaches. Methodology: DNA extraction was carried out by silica columns based total demineralization extraction method.DNA quantification was carried out by Real Time PCR. DNA amplification was carried out by using AmpFlSTR®Identifiler® PCR amplification kit with modified conditions. Capillary electrophoresis and Data analysis were carried out by Genetic Analyzer 3130 (ABI) and GeneMapper ID software version 3.2. Results: DNA was detected in 17 out of 24 skeletal remains. Among them, in 7 samples DNA was in the range of 1-10 pg/µL, in 4 samples it was in the range of 22-69 pg/µL and in 6 samples the DNA was in the range of >100 pg/ µL. The CT value (<30) of 40 cycles indicated that the PCR inhibitors were removed during DNA extraction method. Promising results were obtained by increasing the number of PCR cycles from standard 28 to 33 instead of 32 in PCR reaction. Finally it was observed that consensus approach produced reliable and reproducible DNA profiles from old skeletal remains. Conclusions: Forensic DNA typing of old skeletal remains, through a multiplex AmpFlSTR®Identifiler® PCR amplification kit, is improved by using: a highly effective DNA extraction method, modified and optimized PCR conditions, increasing sensitivity of PCR amplification and consensus approach

Baluchi and Pakhtun population data of 9 X-chromosomal short tandem repeat loci

Baluchistan is the largest province of Pakistan in terms of area, constituting approximately 44% of the countrys total land mass, and the smallest in terms of population, being home to less than 5% of the countrys population. Khyber Pakhtunkhwa (KPK) formerly called North-West Frontier Province is located in the north-west of Pakistan having an estimated 13.4% of total population of Pakistan in which Pakhtuns are the major ethnic group. A total of 250 samples from Baluchi population and 250 samples from Pakhtun population were typed for 9 X-chromosomal STR markers: DXS101, DXS6789, DXS7132, DXS7423, DXS7424, DXS8378, GATA31E08, GATA172D05 and HPRTB along with sex typing locus, Amelogenin. A total of 59 alleles were found in Baluchi population while 61 alleles were found in Pakhtun population. This is the first study of the two populations based on these markers and the population data can be used as reference database for Baluchi and Pakhtun populations.

Scope of X-Chromosomal MiniSTRs: Current Developments

The experience of using autosomal short-length amplicon STRs or miniSTRs in profiling of degraded DNA and mass disaster victims is extended into the realm of X-chromosomal (ChrX) STR miniaturization. About half of the total X-STRs are now short-length amplicons and the focus is shifting to using the mini versions of all of them. Joint multiplexing of these loci can be used for solving complex paternity cases and association of mass disaster victims with their families. This technology may herald a new dimension for research in population genetics and evolution. We present an overview of the progress made thus far and the future scope and prospects for X-miniSTRs.