|Year : 2015 | Volume
| Issue : 1 | Page : 29-35
Genetic diversity between two Igbo men from Owerri senatorial province as determined by autosomal short tandem repeats, Y-chromosomal short tandem repeats and mitochondrial DNA typing methods
Anukam Kingsley Chidozie1, Aliche Isaac2
1 Department of Medical Laboratory Science, TWAS Genomics Research Unit, School of Basic Medical Sciences, University of Benin, Benin, Nigeria
2 Department of Laboratory Services, High Prairie Health Complex, Alberta Health Services, High Prairie, Alberta, Canada
|Date of Web Publication||4-Jun-2015|
Dr. Anukam Kingsley Chidozie
Department of Medical Laboratory Science, TWAS Genomics Research Unit, School of Basic Medical Sciences, University of Benin, Benin
Source of Support: None, Conflict of Interest: None
Background: Human identification has recently been optimized following the completion of human genome sequence, by using DNA markers that exhibit the highest possible variation. However, in developing countries such as Nigeria, the application of DNA typing for identification of human subjects either for forensic or medical purposes is inadequate due to the absence of national forensic DNA laboratories and lack of the legislative framework. Materials and Methods: In this study, two male subjects of Igbo origin provided their blood, hair and buccal samples for DNA analysis. DNA was extracted, purified, quantified with human quantifiler™ polymerase chain reaction reaction mix. Hyper-variable segment 1 of the D-loop mitochondrial DNA (mtDNA HVS-1) was amplified, purified and fragments sequenced and analyzed with ABI Genetic Analyzer. A multiplex AmpFlSTR Identifiler kit amplified 15 STR plus amelogenin loci of the nuclear DNA and 16 STR of the Y-chromosome. GeneMapper ID software version 3.2 was used for the analysis of autosomal and Y-chromosome AmpFlSTR. Results: Result show that the mtDNA lineage of UMBACK subject belongs to L3e2b while ELAMBIA is assigned to L3f1b1 haplogroup. Based on the allelic frequency database, both subjects displayed uniqueness in random match probability for the autosomal allelic short-tandem repeat (STR). Based upon a mutation rate of 0.003 for Y-DNA STR markers, the two individuals most likely shared a common paternal ancestor 96 generations ago. Both subjects were assigned to E1b1a haplogroup with a 100% probability, which is consistent with the haplogroup associated with Igbo people in Nigeria. Conclusion: As expected, well-established forensic genetic tools comprising of mtDNA, autosomal and Y-chromsomomal STR typing methods were all found to distinguish two selected Nigerian Igbo individuals with a very high power of discrimination.
Keywords: Autosomal DNA, DNA typing, Igbo haplogroup, mitochondrial DNA, short tandem repeats
|How to cite this article:|
Chidozie AK, Isaac A. Genetic diversity between two Igbo men from Owerri senatorial province as determined by autosomal short tandem repeats, Y-chromosomal short tandem repeats and mitochondrial DNA typing methods. Niger J Exp Clin Biosci 2015;3:29-35
|How to cite this URL:|
Chidozie AK, Isaac A. Genetic diversity between two Igbo men from Owerri senatorial province as determined by autosomal short tandem repeats, Y-chromosomal short tandem repeats and mitochondrial DNA typing methods. Niger J Exp Clin Biosci [serial online] 2015 [cited 2019 May 21];3:29-35. Available from: http://www.njecbonline.org/text.asp?2015/3/1/29/158164
| Introduction|| |
Recent genetic studies have shown that populations that exhibit high-levels of genetic diversity inhabit the continent of Africa, compared to most other continental populations, and it is thought to be the ancestral home of modern humans.  The frequently observed pattern of reduced genetic diversity away from Africa is seen as strong evidence for the out-of-Africa movement(s) of anatomically modern humans.  African populations have the largest number of population-specific autosomal, X-chromosomal and mitochondrial DNA haplotypes with non-African populations having only a subset of the genetic diversity present in Africa.  Earlier studies have documented that genetic structure of human populations is strongly shaped by the social, cultural and demographic processes that govern migration and settlement of individuals.  In an attempt to provide insight to such population structure differences, most studies analyze genetic differences among widely dispersed human populations. For example, a study revealed strong patterns in the genetic structure of human populations on the small island of Sao Tome that were influenced by spatial-and temporal-specific events. 
In contrast, there is little genetic structure among neighboring ethnic groups in southern Nigeria despite the strong language differentiation among them. 
However, these genetic diversities have been applied in forensic DNA typing, which plays significant roles in protecting the innocent as well as implicating the guilty.  The application of modern DNA typing for identification of individuals appears to be nonexistent in most developing countries, especially in Nigeria. Law enforcement officers still rely on nonscientific physical evidence that may likely convict an innocent person and exonerate the real culprits in crime situations.
The objective of the present study is to use DNA typing methods to screen for a set of 15 autosomal short-tandem repeats (STRs) plus amelogenin, 16 Y-chromosomal STRs and sequencing the hyper-variable segment (HVS-1) of the mitochondrial DNA in order to decipher genetic differences and similarities between two Igbo men from Owerri province.
| Materials and Methods|| |
Sample collection and DNA extraction
The two participants were selected after signed informed consent and based on convenience sampling method. The two subjects were designated as UMBACK and ELAMBIA. They provided three samples (buccal swab, blood, and hair). Blood sample was spotted on FTA™ card (Whatman, UK). DNA extraction was based on the FTA DNA extraction and proteinase k protocols respectively. Briefly, a sterile scissors was used to cut an approximate 0.5 cm × 1.5 cm rectangle from the blood spotted area of the material. The cut section was placed in a sterile 1.5 ml tube and 400 uL of 1X TNE (1.2 mg/ml trizma base, 5.8 mg/ml NaCl, 1 mM ethylene-diamine-tetraacetic acid [EDTA]) buffer was added. After addition of 2 μL of proteinase K (20 mg/ml), the tube was incubated at 56°C overnight with gentle agitation (400 rpm) in the thermomixer. The tube was vortexed for 10 s and centrifuged at 13000 rpm for 1-min. The DNA-containing supernatant was placed in a sterile 2.0 ml tube. The DNA extract was purified with a silica-based commercial DNA QIAquick™ purification kit protocol (Valencia, California, USA).
The DNA from hair was extracted with Guanidine thiocyanate (GuScN) method by adding 500 μL of 4M GuScN (0.1M tris-HCl, 0.02M EDTA, 0.013% Triton X-100, 4GuScN) buffer to the tube containing hair. The tube was incubated overnight at 56°C overnight with gentle agitation in the thermomixer. The DNA extract was purified with a silica bead DNA QIAquick™ purification kit protocol.
DNA from the epithelial cells present in the buccal swab was extracted with chelating resins. The tip of the swab was cut a sterile scissors into a sterile 2.0 mL tube and 400 μL of 10% Chelex buffer was added. The tube was vortexed for 10 s and incubated at 56°C for 3 h with gentle agitation (400 rpm) in the thermomixer. The sample was centrifuged at 13000 rpm for 1.0 min. The supernatant containing DNA was stored at 4°C for a short term or −20°C for a long-term.
DNA quantification with real-time polymerase chain reaction
To determine the amount of amplifiable nuclear DNA in the samples, we prepared the quantification reaction in a 0.2 mL optical stripe tubes comprising 12.5 μL of quantifiler™ polymerase chain reaction (PCR) reaction mix, 10.5 μL of quantifiler™ human primer mix and 2.0 μL of DNA sample, standards and negative control. Eight human genomic DNA quantification standards were obtained from the commercial supplier (Applied Biosytems, Foster City, CA, California, USA) ranging from 50 ng/μL to 0.023 ng/ μL. The quantification mixture was placed on the ABI Prism 7000 thermocycler sequence detection system. The thermocycler was programmed to hold the tubes at 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. The quantity of DNA was detected based on the standard curve produced by the eight standards of housekeeping human telomerase reverse transcriptase gene (hTERT-5p15.33). The TaqMan probe was labeled with a VIC reporter dye and Minor Groove Binder (MGB) quencher dye (Applied Biosystems, Foster City, California, USA). The nuclear DNA (nDNA) primer and probe sequences were used as described by Timken et al.  These target the telomerase gene on chromosome 5p15.33. The nuclear probe is an FAM labeled TaqMan Black Hole Quencher.
| Mitochondrial DNA Polymerase Chain Reaction and Presequence Purification|| |
Polymerase chain reaction for the detection of hyper-variable region 1 of mitochondrial DNA (mtDNA-HVR-1) was done in 25 μL PCR reaction volume comprising of 2.5 μL of 10X PCR buffer, 0.5 μL of 10 mM dNTP mix, 1.0 μL of 50 mM MgCl 2 , 0.25 μL of forward primer (15996F-5¢-CTCCACCATTAGCACCCAAAGC-3¢), 0.25 μL of Reverse primer (16420R-5¢-TGATTTCACGGAGGATGGTG-3'), 15.4 μL of ddH 2 O, 0.1 μL of 5U/mL polymerase and 5 μL of DNA from blood sample. HVS-1 amplification was carried out in a Biorad Mastercycler. The temperature profile for 50 cycles of amplification was hot start (94°C for 2 min), denature (94°C for 30 s), anneal (60°C for 1-min), extend (72°C for 2 min) and finally held at 7°C.
The PCR amplicon was ran on a PAGE for the selection of successful mitochondrial PCR product. The product was purified using silica column purification kit protocol (Applied Biosystems). Briefly, 5 volume of buffer PB was added to 1 volume of the PCR product in the PCR tube. The tube was vortexed, spun down, and the sample was applied to the spin column and centrifuged at 13000 rpm for 1-min. The flow-through was discarded and later placed into the QIAquick™ column with the addition of 700 μL of buffer PE. The spin column was centrifuged at 13000 rpm for 1-min and placed in a sterile 1.5 mL microfuge tube. The DNA sample bound to the column membrane was eluted by adding 30uL of buffer EB (10 mM Tris-Cl, pH 8.5). The spin column was incubated at room temperature for 3 min and centrifuged for 1-min at 13000 rpm. The DNA was stored at 4°C, until required.
Sequencing purified polymerase chain reaction fragment using big dye terminator cycle protocol
Forward and reverse sequencing PCR was prepared in a sterile 0.2 mL reaction tubes containing a total of 10 μL sequencing reaction (0.5 μL of Big Dye Terminator reaction mix-BDT v3.1, 2.0 μL of 5x sequencing buffer, 0.3 μL of 10 μM primer-15996F (5'-CTCCACCATTAGCACCCAAAGC-3') and 16420R (-5'-TGATTTCACGGAGGATGGTG-3') 3.0 μL of purified DNA PCR product, and 4.2 μL of sterile water). A sequencing control sample pGEM (0.5 μL of Big Dye terminator reaction mix-BDT v3.1, 2.0 μL of 5x sequencing buffer, 1.0 μL pGEM, 2.5 μL of sterile water) and 4.0 μL of M13 (-21) primer set (M13-F-5'-CACGACGTTGTAAAACGAC-3'; M13-R-5'-GGATAACAATTTCACACAGG-3') was included. The PCR thermocycler was set at hot start 96°C for 1-min, followed by 15 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s and extension at 60°C for 75 s. Another 5 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s, extension at 60°C for 90 s. Finally 5 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s and extension at 60°C for 2 min. The amplified product was held at 7°C. The sequenced product was purified in order to remove any incorporated dye terminator by using ethanol/sodium acetate solution containing 3.0 μL of 3M sodium acetate, 62.5 μL of 95% ethanol and 24.5 μL of sterile water. The entire sequenced product was transferred into the ethanol/sodium acetate mixture and allowed to sit at room temperature for 20 min. Thereafter the product was centrifuged at maximum speed of 13000 rpm for 20 min. The supernatant was aspirated and discarded. The pellet was mixed with 250 μL of 70% ethanol and vortexed for 30 s and then centrifuged at 13000 rpm for 5 min. The supernatant was carefully aspirated to remove as much liquid as possible. The sample was finally dried in a vacuum centrifuge for 15 min.
Loading sample into the ABI3130xl Genetic Analyzer
In the tube containing the dried sample, 15 μL of Hi-Di Formamide was added, vortexed for 1-min and briefly spun down. The sample tube was heated at 95°C for 3 min and immediately chilled on ice for 2 min. The sample was vortexed and spun down and the entire 15 μL sample pipetted into the assigned well in the ABI plate. The ABI3130xl was run on sequencing_36 cmPOP4 protocol and analyzed with 3130POP4_BDTv3-DB.
Short tandem repeats multiplex polymerase chain reaction for autosomal DNA and fragment analysis loading
A multiple PCR for STR was prepared using the forensic AmpFlSTR Identifiler Plus STR kit from Applied Biosystems (Foster City, California, USA). The kit co-amplifies the repeat regions of 15 STR loci, plus amelogenin (X and Y chromosome segment). A positive 10.0 μL quantified DNA blood sample containing 1.0 ng of DNA was added to a 0.2 mL PCR tube, followed by 10 μL of AmpFlSTR Identifiler Plus™ master mix, and 5.0 μL of AmpFlSTR Plus™ primer mix. Positive control (9947A) and PCR negative control were also included. Amplification temperatures in a Biorad master cycler were set at 95°C for 11 min, followed by 28 cycles of 94°C for 20 s, 59°C for 3 min and final extension at 60°C for 10 min. Amplified sample tube was vortexed and spun down. Into a 9.3 μL of master mix containing 0.3 μL of internal size standard (MRL 500) and 9.0 μL of Hi-Di Formamide, 1.0 μL of the amplified sample was added. Allelic ladder (1.0 μL) was run in parallel with the sample and positive control. The sample mixture was denatured at 95°C for 3 min and immediately chilled on ice for 2 min. The sample was vortexed briefly, spun down and loaded into the ABI plate and analyzed on the 16-capillary ABI Genetic Analyzer 3130xl (Applied Biosystems, Foster City, California), which was ran on FA_G5_35 cmPOP4 (Performance Optimized Polymer) protocol.
Multiplex polymerase chain reaction for Y-Chromosomal analysis
A reaction tube (0.2 mL) containing 9.2 μL of AmpFlSTR Y-filer reaction mix, 5.0 μL of AmpFlSTR Y-filer primer mix was set up. This was followed by addition of 0.8 μL of Taq polymerase, and a positive 10.0 μL quantified DNA blood sample containing 1.0 ng of DNA. The PCR master cycler temperature followed a hot start at 95°C for 11 min and a 30 cycles of denature (94°C for 1-min), anneal (61°C for 1-min), extension (72°C for 1-min) and final extension (60°C for 80 min) and thereafter held at 7°C. Amplified sample tube was vortexed and spun down. Into a 9.3 μL of master mix containing 0.3 μL of size standard (MRL500) and 9.0 μL of Hi-Di Formamide, 1.0 μL of the amplified sample was added. Allelic ladder (1.0 μL) was run in parallel with the sample and male positive control (SF-007+) of European origin. The sample mixture was denatured at 95°C for 3 min and immediately chilled on ice for 2 min. The sample was vortexed briefly, spun down and loaded into the ABI plate and ABI Genetic Analyzer.
Analysis of sequence data
The quality of the sequences generated by automated DNA sequencing system (Applied Biosystems 3130xl, Foster City, California) was checked, and nucleotide polymorphisms were analyzed by Sequencher 4.0.5 software (Genecodes, Ann Arbor, MI). Nucleotide changes were noted in comparison with the revised Cambridge Reference Sequence (rCRS). , Assignment of sequences to specific haplogroups was performed according to criteria of Lee et al. 
The mitochondrial DNA sequence generated from the ABI Genetic Analyzer was compared with the rCRS showing the D-loop region 15961-840. Using the online mitochondrial DNA manager (www.mtmanager.yonsei.ac.kr)  as a forensic mtDNA search tool, we searched for mutation matches on control regions (np 16024-16569) compared with the rCRS. GeneMapper ID Software™ version 3.2 (Applied Biosystems, Foster City, California) was used specifically for the analysis of autosomal and Y-chromosome AmpFlSTR samples.
The African-American allelic frequency database,  (available online www.astm.org) for the autosomal STR markers, was used to calculate the Random Match Probability. PopAffiliator 2  was used to assign the subjects to major population groups (http://cracs.fc.up.pt/~nf/popaffiliator2/). Whit Athey's Haplogroup predictor algorithm  was used for the prediction of Y-chromosomal haplogroup. We used the algorithm developed by Walsh  to calculate the probabilities of sharing a common paternal ancestor based on a comparison of the number of Y-DNA STR markers that they share in common versus the number of Y-DNA STR markers that were tested.
| Results|| |
The result of the RT-PCR DNA quantification was based on the normalized standards indicating the buccal, blood, and hair samples from UMBACK had 3.40 ng/uL, 10.42 ng/uL, 0.097 ng/uL of DNA concentrations respectively, while Buccal, Blood, hair from ELAMBIA had 3.17 ng/uL, 16.2 ng/uL, 33.74 ng/uL of DNA. The hair sample from ELAMBIA produced DNA yields more than 300-fold higher than that from UMBACK due to the difference observed in the collection of the hair that included the hair root and the quantity of blood that was spotted on the FTA card. However, quantified DNA from the blood sample was used for the DNA typing of both subjects.
[Table 1] shows the mtDNA mutation polymorphism profiles for UMBACK in relation to rCRS as 16,172C [Figure 1], 16,189C, 16,223T, 16,320T, and ELAMBIA had the following polymorphism profiles 16,129A [Figure 2], 16,209C, 16,223T, 16,292T, 16,295T, and 16,311T. The search on www.mtmanager.yonsei.ac.kr shows that UMBACK mtDNA haplotype profile matched 1148 target samples in the FBI-African database that belongs to L3e2b haplogroup. This group falls within the metapopulation and subpopulation as Africans. The search shows that ELAMBIA mtDNA haplotype profile matched 248 target samples in the FBI-African database that belongs to L3f1b1 haplogroup. This group also falls within the metapopulation and subpopulation as Africans.
|Figure 1: Electropherogram showing the mutation polymorphism for UMBACK mtDNA at position #16172C (cytosine substitution) in relation to the revised Cambridge Reference Sequence at position 16,172T (thymine)|
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|Figure 2: Electropherogram showing the mutation polymorphism for ELAMBIA mtDNA at position #16,129A (adenine substitution) in relation to the revised Cambridge Reference Sequence at position 16,129G (guanine)|
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Autosomal STR markers for the 16 STR co-amplified showed that UMBACK has three homozygous allelic markers occurring at CSF1PO (5q33.1) with 12 repeats (AGAT), D3S1358 (3q21.31) with 15 repeats and D13S317 (13q31.1) with 12 repeats. Heterozygous allelic repeats were observed on the other 13 STR markers as represented on D2S1338 (18, 22 repeats) 12 and 13 repeats on D19S433 and 16, 17 repeats on vWA [Figure 3]. ELAMBIA has two homozygous allelic markers at D13S317 (13q31.1) with 12 repeats (TATC) and vWA (12q13.31) with 19 repeats.
|Figure 3: Autosomal short-tandem repeat identifier plus panel electropherogram showing the heterozygous repeats observed for UMBACK at D2S1338 (18, 22 repeats) DS19433 (12, 13 repeats) and vWA (16, 17 repeats)|
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Using the African-American allelic frequency database  for the STR markers, we calculated the random match probability for UMBACK (3.415E+18) and for ELAMBIA (7.713E+19). With the allelic STR repeats, UMBACK was assigned to Sub-Saharan Africa with a probability of 75.5%, Eurasia 12.6% and Asia 11.9%. Surprisingly, ELAMBIA's allelic STR repeats was assigned to Sub-Saharan Africa with 71% probability, Eurasia 22.8% and Asia 6.2%.
Y-chromosome STR markers shown in [Figure 4] indicates that out of the 16 STR markers tested, both subjects have 9 (56.25%) similarities in the allelic repeats comprising DYS389I, DYS390, DYS391, DYS439, DYS635, DYS392, YGATAH4, DYS437, and DYS438. We used the algorithm developed by Walsh  to calculate the probabilities that UMBACK and ELAMBIA shared a common paternal ancestor based on a comparison of the number of Y-DNA STR markers that they share in common versus the number of Y-DNA STR markers that were tested and compared between the two individuals. Match at 9 out of 16 Y-DNA STR markers revealed that there is a 50% chance that the two individuals shared a common paternal ancestor within the last 106 generations. Based upon a mutation rate of 0.003 for Y-DNA STR markers, the two individuals most likely shared a common paternal ancestor 96 generations ago [Figure 5]. Whit Athey's Haplogroup predictor algorithm was used for the prediction of Y-chromosome haplogroup as shown in [Table 2]. UMBACK was found to belong to E1b1a with a fitness score of 72 and 100% probability, while ELAMBIA also belonged to the same haplogroup (100% probability) with less fitness score of 32.
|Figure 4: Y-chromosome short-tandem repeat (STR) markers showing that out of the 16 STR markers tested, both subjects have 9 (56.25%) similarities in the allelic repeats comprising DYS389I, DYS390, DYS391, DYS439, DYS635, DYS392, YGATAH4, DYS437, and DYS438. POS-Y QC is the positive Y-chromosome sample|
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|Figure 5: Posterior distribution or Likelihood and cumulative probability showing Match at 9 out of 16 Y-DNA short-tandem repeat (STR) markers, based upon a mutation rate of 0.003 for Y-DNA STR markers|
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|Table 2: Y-chromosomal haplogroup prediction using whit Athey's algorithm|
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| Discussion|| |
We relied on the unique features of mtDNA that are involved in maternal inheritance, high mutation rate and absence of recombination to analyze the two subjects. The identified mutations or polymorphism in the two subjects in comparison to the rCRS shows that they could not have shared a common maternal ancestry. The collective polymorphism profile of UMBACK haplotype indicated that his mtDNA haplogroup - a specific genetic population to which he belongs is L3e2b. This group is associated with mtDNA D-loop HVS-1 sequence motif (172-189-223-320). The L3e clade is the most widespread, frequent, and ancient of the African L3 clades, comprising approximately one-third of all L3 types in sub-Saharan Africa.  This haplogroup has recently been discussed in detail by Bandelt et al.  who suggested an origin for the haplogroup to be in the Central Africa/Sudan region ~45,000 years ago. The subclade L3e2b is found primarily in West Africa, This indicates a range expansion from Central into West Africa (~9,000 years ago).
In contrast, ELAMBIA's mtDNA haplotype belongs to L3f1b1 haplogroup. This group is associated with HVS-1 sequence motif (209-223-311). This haplogroup is also shared by West and South-East Africans. At the same time, several L3f types are shared uniquely by West Africans only. L3f is likely of East African origin according to Salas et al.  but the derived sub-haplogroup L3f1 is also present in West Africa, and it is this component that is most commonly found in Americans. It is thought that L3f1b1 lineage to which ELAMBIA belongs is dated to 9, 200 years, while UMBACK L3e2b lineage arose earlier approximately 9,000 years.  However, studies of both the mitochondrial DNA (mtDNA) mismatch patterns in modern African populations and related mtDNA lineage-analysis patterns point to a major demographic expansion centered broadly within the time range from 80,000 to 60,000 before present, probably deriving from a small geographical region of Africa.  Some authors have argued that natural selection may have played a role in shaping human regional mtDNA variation and that one of the selective influences was climate.  It may be possible that the variance observed between UMBACK and ELAMBIA in their mtDNA hyper-variable region 1, could be due to different maternal lineage over a long period.
The autosomal STR markers provided another major diversity between UMBACK and ELAMBIA. The Random Match Probability for UMBACK (3.415E + 18) and for ELAMBIA (7.713E + 19) suggests that they have a very high power of discrimination. This high power of discrimination is also highlighted by the difference observed in the population assignment by popaffiliator 2 algorithm. The only common allelic STR marker with 12 homozygous repeats, which they had was noted on D13S317. This STR locus is characterized by TATC repeat sequence and 7-15 alleles are known to exist with the highest frequency of over 80% occurring with 12 alleles. , African genetic diversity and its positive correlation with both geography and language has previously been well described at the continent-wide scale for both uniparental and autosomal markers. 
The Y-chromosome STR markers assigned both subjects to E1b1a haplogroup using Whit Athey's Haplogroup predictor. Haplogroup E1b1, which is characterized by a high degree of internal diversity, is the most represented Y chromosome haplogroup in Africa. Recent study has shown that haplogroup E1b1 now contains two basal branches, E-V38 (E1b1a) and E-M215 (E1b1b), with V38/V100 joining the two previously separated lineages E-M2 (former E1b1a) and E-M329 (former E1b1c). E-M2 is the most common haplogroup in sub-Saharan Africa, with frequency peaks in West (about 80%) and Central Africa (about 60%). 
| Conclusion|| |
In summary, this study has demonstrated that two individuals from the same ethnic/language group can be separated using the single nucleotide polymorphisms of the mtDNA D-loop HVS-1 profile. The two subjects did not have a common maternal lineage, therefore making it imperative for a need for population study in Nigeria in order to determine how rare/common any given mtDNA profile would be. The autosomal STR markers provided a very high power of discrimination, which has potential application for identifying individuals in any forensic and or medical situation. Interestingly, the Y-DNA STR markers, linked the two individuals that they most likely shared a common paternal ancestor 96 generations ago.
| Acknowledgments|| |
The co-operation of Stephen Fratpietro, The Manager of Paleo DNA Laboratory, Lakehead University, Thunder Bay, ON, Canada, for excellent training and use of their facility is valued in great measure.
The approval given to Isaac Aliche by Angela Mutter, Satinder Walia, Wanda June, Wendy Corbiere and Tammy Hofer all from High Prairie Health complex is appreciated.
The encouragement and support to Kingsley from Ruth McManus of Endocrinology Department, St. Joseph's Hospital, London, ON is also highly appreciated.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]