Sequence polymorphism of the mitochondrial DNA control region in the population of Vojvodina Province,Serbia

In order to generate and establish the database for forensic identification purposes in Vojvodina Province (Serbia), the sequence of the hypervariable regions 1 (HV1) and 2 (HV2) of the mtDNA control region were determined in a population of 104 unrelated individuals from Vojvodina Province, using a fluorescent-based capillary electrophoresis sequencing method. A total of 93 different haplotypes were found, of these 83 mtDNA types were unique, nine haplotypes were shared by two individuals and one haplotype by three individuals. The variation of mtDNA HV1 and HV2 regions was confined to 116 nucleotide positions, of which 72 were observed in the HV1 and 44 in the HV2. A statistical estimate of the results for this population showed the genetic diversity of 0.9977 and the random match probability of 1.18%. Haplogroup H was the most common haplogroup (43.3%). Haplogroups observed at intermediate levels included clusters U (13.5%), T (10.6%), J (8.6%) and W (5.8%).     

A new database of mitochondrial DNA hypervariable regions I and II sequences from 162 Japanese individuals

A database of mitochondrial DNA (mtDNA) hypervariable region 1 (HV1) and region 2 (HV2) sequences of the mtDNA control region was established from 162 unrelated Japanese individuals. The random match probability and the genetic diversity for this database were 0.96% and 0.997, respectively. Length heteroplasmy in the C-stretch regions located around position 16189 in HV1 and 310 in HV2 was observed in 37% and 38% of the samples, respectively. A strategy using internal sequencing primers was devised to obtain confirmed sequences in these length heteroplasmic individuals. This database, combined with other mtDNA sequence databases from the Japanese population, will permit the significance of mtDNA match results to be properly reported in mtDNA typing casework in Japan.Received: 10 August 2000 / Accepted: 7 January 2001An erratum to this article can be found at     

Sequence polymorphism of the mitochondrial DNA control region in the Slovenian population

The forensic application of mitochondrial DNA (mtDNA) typing requires large and regionally well-defined databases. To expand the database for forensic identification purposes in Slovenia, the mtDNA control region sequences of the hypervariable regions HVI and HVII were determined in a population of 129 maternally unrelated Slovenians, using a fluorescent-based capillary electrophoresis sequencing method. A total of 111 different haplotypes resulting from 124 polymorphic positions (80 polymorphic positions in HVI and 44 in HVII) were found. Of these, 101 mtDNA types were unique, 6 haplotypes were shared by 2 individuals, 1 haplotype by 3 individuals, 2 haplotypes by 4 individuals, and the most common haplotype was found in 5 individuals. The most frequent haplotypes in the Slovenian population ,263(G), 315.1(C) and 263(G), 309.1(C), 315.1(C) are also the most common in other European populations. The data support the concept that these haplotypes may represent a common European mtDNA sequence types. The sequence poymorphisms were compared to the databases of west Austria and central Italy and the HVI and HVII sequence matching probabilities within and between populations were calculated. It is 1.1–4.5 times more likely to find a sequence match in a random pair of Slovenians than in a random Slovenian-Italian pair and in a random Slovenian-Austrian pair. The length heteroplasmy in the homopolymeric C-stretch regions located at nucleotide positions 16184–16193 in HVI and at positions 303–315 in HVII was observed in 17% and 8% of individuals, respectively. A statistical estimate of the results for this population showed the random match probability and the genetic diversity of 1.16% and 0.996, respectively.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00414-003-0394-3     

Mitochondrial DNA sequences for 118 individuals from northeastern Spain

A population database was generated from 118 unrelated Caucasoid individuals living in Spain. Sequence polymorphisms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II (HVRI and HVRII) were determined using the polymerase chain reaction (PCR) and direct sequencing. A total of 102 different sequences were found as defined by 105 variable positions. The most common sequence occurred six times, and this sequence is also the most frequent in other European populations such as Austria, Germany and Britain. The mean pair-wise difference for the two HVR regions taken together was 7.74. The study revealed that transitions made up the majority of the variations (88%), whereas we observed a significantly lower frequency of transversions (8%). Also one individual in this study was observed with two positions of heteroplasmy at nucleotides 150 (C/T) and 153 (G/A). A statistical estimate of the results for this population showed a genetic diversity of 0.99. The probability of two random individuals showing identical mtDNA haplotypes is 1.3%. In order to use the mtDNA analysis in forensic casework, we consider that it is of crucial importance to know the frequency of the different sequences of mtDNA, and this data base study could be a useful tool to statistically evaluate the results. Received: 12 July 1999 / Accepted: 11 April 2000     

Expanding the forensic German mitochondrial DNA control region database: genetic diversity as a function of sample size and microgeography

Mitochondrial DNA control region sequences were determined in 109 unrelated German Caucasoid individuals from north west Germany for both hypervariable regions 1 (HV1) and 2 (HV2) and 100 polymorphic nucleotide positions (nps) were found, 63 in HV1 and 37 in HV2. A total of 100 different mtDNA lineages was revealed, of which 7 were shared by 2 individuals and 1 by 3 individuals. The probability of drawing a HV1 sequence match within the north west Germans or within published sets of south Germans and west Austrians is similar (within a factor of 2) to drawing a sequence match between any two of these three population samples. Furthermore, HV1 sequences of 700 male inhabitants of one village in Lower Saxony were generated and these showed a nearly linear increase of the number of different haplotypes with increasing number of individuals, demonstrating that the commonly used haplotype diversity measure (Nei 1987) for population samples tends to underestimate mtDNA diversity in the actual population. Received: 14 January 1999 / Accepted: 15 February 1999     

Haplotype diversity in human mitochondrial DNA hypervariable regions I–III in the city of Caracas (Venezuela)

In order to expand the database of variable DNA for forensic identification purposes in Venezuela, we analyzed the sequence polymorphisms of mitochondrial DNA (mtDNA) hypervariable regions (HVR) I–III from 100 unrelated individuals from the city of Caracas, using PCR amplification and fluorescent-based capillary electrophoresis sequencing method. Dominant haplogroups corresponded to Native Americans followed by African ones. The inclusion of HVR III although useful for sub-haplogroup assignation, added little to the discrimination capacity of our database.     

Location and frequency of polymorphic positions in the mtDNA control region of individuals from Germany

In order to identify polymorphic positions and to determine their frequency in the human mitochondrial D-loop containing region, the mitochondrial DNA (mtDNA) control region of 200 unrelated individuals from Germany were amplified and directly sequenced. Sequence comparison led to the identification of 190 mitochondrial lineages as defined by 202 variable positions. The most frequently occurring lineage comprised 5 individuals, whereas 186 types of D-loop sequences were observed in only one individual. Of the sequences studied 7% are not unique but show at least one counterpart with an identical haplotype. The majority (61%) of the control regions investigated showed between four and eight nucleotide positions deviating from the reference sequence. The maximum number of deviations observed in a single control region was 18. The majority of the variable positions in the D-loop region (88%) are located within three hypervariable regions. Sequence variations are caused by nucleotide substitutions, insertions or deletions. As compared to insertions and deletions, nucleotide substitutions make up the vast majority of the mutations (90%). We have predominantly found transitions (75%) and a significantly lower frequency of transversions (15%) whereas insertions (6%) as well as deletions (4%) are rather rare. Upon sequencing the mitochondrial control region from 200 German Caucasians the genetic diversity was estimated at 0.99. The probability of two randomly selected individuals from a population having identical mtDNA types is 0.6%. Received: 21 May 1997 / Received in revised form: 9 September 1997     

Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics

Resultsof sequencing of whole mitochondrial genome, HV1 and HV2 DNA with the second generation system (SGS) Roche 454 GS Junior were compared with results of Sanger sequencing and SNP typing with SNaPshot single base extension detected with MALDI-TOF and capillary electrophoresis. We investigated the performance of the software analysis of the data, reproducibility, ability to sequence homopolymeric regions, detection of mixtures and heteroplasmy as well as the implications of the depth of coverage. We found full reproducibility between samples sequenced twice with SGS. We found close to full concordance between the mtDNA sequences of 26 samples obtained with (1) the 454 SGS method using a depth of coverage above 100 and (2) Sanger sequencing and SNP typing. The discrepancies were primarily observed in homopolymeric regions. The 454 SGS method was able to sequence 95% of the reads correctly in homopolymers up to 4 bases, and up to 6 bases could be sequenced with similar success if the results were carefully, visually inspected. The 454 technology was able to detect mixtures or heteroplasmy of approximately 10%. We detected previously unreported heteroplasmy in the GM9947A component of the NIST human mitochondrial DNA SRM-2392 standard reference material.     

HVI and HVII mitochondrial DNA data in Apaches and Navajos

Most mtDNA studies on Native Americans have concentrated on hypervariable region I (HVI) sequence data. Mitochondrial DNA haplotype data from hypervariable regions I and II (HVI and HVII) have been compiled from Apaches (N=180) and Navajos (N=146). The inclusion of HVII data increases the amount of information that can be obtained from low diversity population groups. Less mtDNA variation was observed in the Apaches and Navajos than in major population groups. The majority of the mtDNA sequences were observed more than once; only 17.8% (32/180) of the Apache sequences and 25.8% of the Navajo sequences were observed once. Most of the haplotypes in Apaches and Navajos fall into the A and B haplogroups. Although a limited number of haplogroups were observed, both sample populations exhibit sufficient variation for forensic mtDNA typing. Genetic diversity was 0.930 in the Apache sample and 0.963 in the Navajo sample. The random match probability was 7.48% in the Apache sample and 4.40% in the Navajo sample. The average number of nucleotide differences between individuals in a database is 9.0 in the Navajo sample and 7.7 in the Apache sample. The data demonstrate that mtDNA sequencing can be informative in forensic cases where Native American population data are used.     

Sequence polymorphism in the mtDNA HV1 region in Japanese and Chinese

We investigated the nucleotide substitution and insertion/deletion polymorphism of the HV1 region in mtDNA by sequencing blood samples from 150 unrelated Japanese and 120 unrelated Chinese and revealed 108 sequence types from the Japanese group and 87 sequence types from the Chinese. Some substitutions were characteristic of East Asian populations as compared with data reported on Caucasian populations, and some were area-specific among East Asians. The level of genetic diversity and genetic identity revealed by this system was superior to that obtained by VNTR systems for nuclear DNA. These results show the usefulness of mtDNA sequencing in forensic examination for individual identification. We also found some sequence variations in the homopolymeric tract of cytosine (np16180-16194 in the Anderson's reference sequence) that might suggest some hints regarding the mechanisms for and the development of heteroplasmic length variations in this tract.     

Mitochondrial DNA sequence variation in Jewish populations

Sequence analysis of HVRI and HVRII mitochondrial DNA was carried out on 107 Jewish samples from Ashkenazi, Oriental, North African, and Sephardic origins. Control region sequences were assigned to a haplogroup by means of the analysis of the RFLP motif -7025 AluI or by using sequence motifs. A total of 88 different haplotypes were observed with a lower incidence of unique haplotypes (68.2%) than in other populations. Four individuals with one position of sequence heteroplasmy at nucleotides 16093, 16134, 16169, and 235, respectively, were detected. The mean pairwise difference in the Jewish population was 9.7 nucleotides. The gene diversity was 0.996, and the random match probability was 1.3%. When the data were compared with the autosomal and Y-chromosome markers previously studied in these populations, sex-specific differences could be observed in the Jewish populations. This fact must be taken into account for choosing suitable databases to correctly weigh the value of the evidence of a mtDNA and/or Y profile match.Electronic Supplementary Material Supplementary material, including all sequencing electropherograms, is available for this article at     

Subtyping mtDNA haplogroup H by SNaPshot minisequencing and its application in forensic individual identification

Sequence variation of the hypervariable segments (HVS) I/II of mitochondrial DNA (mtDNA) and the haplogroup affiliation were determined in a sample of 271 Italian subjects. This analysis showed that 42% of the individuals could be ascribed to H, the most frequent haplogroup in European Caucasian populations. This fraction was then screened for specific single nucleotide polymorphisms located in the coding region to identify H subclades H1–H15. We set up two multiplex polymerase chain reactions and specific SNaPshot assays to investigate the frequency distribution of these subgroups in our population sample and to examine their usefulness in discriminating among commonly shared HVS I/II sequences. This allowed the assignment of a large portion of the mtDNAs (∼70%) to specific subhaplogroups, with H1 and H5 being the most represented. About two-thirds of the individuals sharing common HVS I/II sequences were subdivided and ascribed to specific H subhaplogroups with a significant reduction of the frequencies of the most common mtDNA haplotypes. Haplogroup H subtyping could thus be extremely useful in forensic identification when many samples have to be analysed and compared, avoiding excessive time-consuming and labor-intensive sequencing analysis.     

Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: Application of mtDNA sequence analysis to a forensic case

The sequence of the two hypervariable segments of the mitochondrial DNA (mtDNA) control region was generated for 101 random Austrian Caucasians. A total of 86 different mtDNA sequences was observed, where 11 sequences were shared by more than 1 individual, 7 sequences were shared by 2 individuals and 4 sequences were shared by 3 individuals. One of the four most common mtDNA sequences in Austrians is also the most common sequence in both U.S. and British Caucasians, found in approximately 3.0% of Austrians, 4.0% of British, and 3.9% of U.S. Caucasians. Of the remaining three common Austrian sequences, one was not observed in either U.S. or British Caucasians. However, three British Caucasians exhibited a similar sequence type. Therefore, this particular cluster of sequence polymorphisms may represent a common “European” mtDNA sequence type. In general, Austrian Caucasians show little deviation from other Caucasian databases of European descent. Finally, mtDNA sequence analysis was applied to a forensic case, where hairs found at a crime scene matched the control hairs from the suspect. Received: 30 June 1997 / Received in revised form: 12 November 1997     

Phylogenetic classification of Japanese mtDNA assisted by complete mitochondrial DNA sequences

We investigated control and coding region polymorphisms in mitochondrial DNA (mtDNA) in 100 unrelated individuals from a Japanese population and determined the basal phylogenetic haplogroup lineages in all samples under updated information. Many of the basal phylogenetic haplogroup lineages assigned on East Asian mtDNA haplogroups corresponded to those previously established. However, new haplogroup lineages such as M7a2a, M7a2b, M7a2*, M7c1b, M11b2*, G2b*, D4c1b1a, D4g2b, A4*, A9, N9b*, B4d1, B4d2, and F1e were identified and established by complete sequencing. Although sequence comparison of the 1.15-kb control region identified 84 mitochondrial haplotypes, examination of coding region polymorphisms increased the total number of haplotypes to 91. Determination of the basal haplogroup lineages increased the discrimination power of mtDNA polymorphisms for personal identification and their usefulness in determining geographic origin in forensic casework in Japanese and other East Asian populations.     

Investigation of length heteroplasmy in mitochondrial DNA control region by massively parallel sequencing

Accurate sequencing of the control region of the mitochondrial genome is notoriously difficult due to the presence of polycytosine bases, termed C-tracts. The precise number of bases that constitute a C-tract and the bases beyond the poly cytosines may not be accurately defined when analyzing Sanger sequencing data separated by capillary electrophoresis. Massively parallel sequencing has the potential to resolve such poor definition and provides the opportunity to discover variants due to length heteroplasmy. In this study, the control region of mitochondrial genomes from 20 samples was sequenced using both standard Sanger methods with separation by capillary electrophoresis and also using massively parallel DNA sequencing technology. After comparison of the two sets of generated sequence, with the exception of the C-tracts where length heteroplasmy was observed, all sequences were concordant. Sequences of three segments 16184–16193, 303–315 and 568–573 with C-tracts in HVI, II and III can be clearly defined from the massively parallel sequencing data using the program SEQ Mapper. Multiple sequence variants were observed in the length of C-tracts longer than 7 bases. Our report illustrates the accurate designation of all the length variants leading to heteroplasmy in the control region of the mitochondrial genome that can be determined by SEQ Mapper based on data generated by massively parallel DNA sequencing.     

Mitochondrial DNA in human hair shafts – existence of intra-individual differences?

The sequences of the hypervariable region 1 (HV1) of the mitochondrial DNA control region from multiple hair shafts from 10 unrelated individuals were compared to determine the frequency of differences in hairs from one individual. The extraction method described herein showed an average success rate of 67% for all 150 hair shafts tested in HV1. The mtDNA sequences from the hair shafts matched the sequences from the corresponding blood and saliva samples taken from the same donor and no evidence of heteroplasmy was found. The results emphasize the reliability of DNA extraction and mtDNA typing from human hair shafts for forensic purposes. Received: 16 July 1998 / Received in revised form: 31 August 1998     

Pilot study for forensic evaluations of the Precision ID GlobalFiler™ NGS STR Panel v2 with the Ion S5™ system

With the continuous development of massively parallel sequencing (MPS), increasing numbers of laboratories have utilized this method for forensic genomic analyses. When sequencing common short tandem repeats (STRs), MPS does have many advantages over the length-based genotyping method that uses traditional capillary electrophoresis (CE) technology. The Precision ID GlobalFiler™ NGS STR Panel v2 was recently released to simultaneously target 31 autosomal STRs (20 expanded Combined DNA Index System (CODIS) core loci and 11 non-CODIS loci) and 4 gender determination loci (Amelogenin, DYS391, SRY and Y-indel (rs2032678)) with the Ion S5™ System. In the current study, we performed a preliminary validation for this novel MPS-STR panel that included the following analyses: repeatability, concordance, stutter and balance, sensitivity, case-type sample testing, stability, mixture and a population investigation. Complete and reliable profiles were obtained using 125 pg of positive control DNA. The commonly encountered types of case samples and artificial mixtures with ratios of 1:1, 1:3 and 3:1 were also fully genotyped. Additional allele sequence variations were detected in samples from 50 unrelated individuals, and subsequently, an increased power of discrimination and power of exclusion were achieved. However, the average depth of coverage (DoC) of the Penta D locus was detected to be dramatically lower than those of other loci, which caused an interlocus imbalance; this could be one of the reasons for the intralocus imbalance of this locus and the 0.18% inconsistent results in the concordance study. Although certain flaws were observed, the informative metrics, including the DoC, sequence coverage ratios (SCRs) and heterozygote balance (H_b), of the novel MPS multiplex in our study were sufficient for reliable sequencing results that were 99.61% in concordance with the capillary electrophoresis (CE) results. In general, the Precision ID GlobalFiler™ NGS STR Panel v2 was demonstrated to be sensitive, reliable and robust and could be a powerful tool for human identification and kinship analyses. Additionally, we look forward to its updated version.     

Sequence polymorphism of mitochondrial DNA in Japanese individuals from Gifu Prefecture

Sequence polymorphisms of the hypervariable region HV1 in mitochondrial DNA (mtDNA) were analyzed in a sample of 137 unrelated Japanese individuals living in Gifu Prefecture (central region of Japan) using polymerase chain reaction amplification and direct sequencing. Eighty-two different haplotypes resulting from 81 variable sites were found in the mtDNA HV1 region between positions 16061 and 16450. The most frequent haplotype (16223T, 16362C) was shared by ten individuals. The genetic diversity and the genetic identity were 0.985 and 0.022, respectively. The C-stretch region located around position 16189 was observed in 23.4% of this population sample. Sequence heteroplasmy at the position 16103 (A/G) was found in one individual.     

Belgian canine population and purebred study for forensics by improved mitochondrial DNA sequencing

In canine population studies for forensics, the mitochondrial DNA is profiled by sequencing the two hyper variable regions, HV1 and HV2 of the control region. In a first effort to create a Belgian population database some samples showed partially poor sequence quality. We demonstrated that a nuclear pseudogene was co-amplified with the mtDNA control region. Using a new combination of primers this adverse result was no longer observed and sequencing quality was improved. All former samples with poor sequence data were reanalyzed. Furthermore, the forensic canine population study was extended to 208 breed and mixed dogs. In total, 58 haplotypes were identified, resulting in an exclusion capacity of 0.92. The profile distribution of the Belgian population sample was not significantly different from those observed in population studies of three other countries. In addition to the total population study 107 Belgian registered pedigree dogs of six breeds were profiled. Per breed, the obtained haplotypes were supplemented with those from population and purebred studies. The combined data revealed that some haplotypes were more or less prominent present in particular dog breeds. The statistically significant differences in haplotype distribution between breeds and population sample can have consequences on mtDNA databasing and matching probabilities in forensics.     

Sequence-based allelic variations and frequencies for 22 autosomal STR loci in the Lebanese population

This is the first study that characterizes the sequence-based allelic variations of 22 autosomal Short Tandem Repeat (aSTR) loci in a population dataset collected from Lebanon. Genomic DNA extracts from 195 unrelated Lebanese individuals were amplified with PowerSeq 46GY System Prototype. Targeted amplicons were subjected to DNA library preparation and sequenced on the Verogen MiSeq FGx Sequencing System. Raw FASTQ data files were processed by STRait Razor v3. Sequence strings were annotated according to the considerations of the DNA Commission of the International Society for Forensic Genetics (ISFG) and tabulated herein with their respective allelic frequencies and GeneBank accession and version numbers. The sequenced Lebanese dataset resulted in 429 distinct allelic sequences as compared to the 236 alleles identified by length only. The increase in the number of alleles was observed at 18 out of 22 aSTR loci and was attributed to the sequence variations residing in both the STR repeat motifs and flanking regions. The study uncovered 25 novel aSTR allelic sequences across 12 loci for which GenBank records did not previously exist in the STRSeq BioProject, PRJNA380127. For a concordance check, the length-based allelic calls derived from the full sequences were compared to those genotyped using capillary electrophoresis (CE) methods. Population genetic parameters relevant to the evaluation of forensic DNA evidence were assessed for the sequence-based data and compared to the parameters generated from the length-based information. Using the sequence-based data, Analysis of MOlecular VAriance (AMOVA), genetic distances, and population genetic structure were evaluated for 1231 individuals sampled from the Lebanese and four U.S. populations (African American, Asian, Caucasian, and Hispanic). The results were tabulated and visualized in a population tree, multidimensional scaling scatter plots, and bar plots. This newly established sequence-based database for the Lebanese population can be beneficial for extending NGS applicability to casework or paternity testing and assessing the strength of evidence for NGS-STR profiles. The described novel sequence variants at certain loci can further help in the effort to characterize the sequence diversity of STR markers from different populations around the world.