Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209–16,400) and B- (positions 30–284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.     

Development and validation of a SYBR green-based mitochondrial DNA quantification method by following the MIQE and other guidelines

In forensic mitochondrial DNA (mtDNA) analysis, quantitative PCR (qPCR) is usually performed to obtain high-quality sequence data for subsequent Sanger or massively parallel sequencing. Unlike methods for nuclear DNA quantification using qPCR, a calibrator is necessary to obtain mtDNA concentrations (i.e., copies/µL). Herein, we developed and validated a mtDNA quantification method based on a SYBR Green assay by following MIQE [Bustin et al., Clin. Chem. 55 (2009) 611-22] and other guidelines. Primers were designed to amplify nucleotide positions 16,190–16,420 in hypervariable region 1 for qPCR using PowerUp SYBR Green and QuantStudio 5. The optimized conditions were 0.3 µM each primer and an annealing temperature of 60 °C under a 2-step cycling protocol. K562 DNA at 100 pg/µL was converted into a mtDNA concentration of 16,400 copies/µL using linearized plasmid DNA. This mtDNA calibrator was obtained by cloning the synthesized DNA fragments of mtDNA (positions 16,140–16,470) containing a 100-bp inversion. The linear dynamic range of the K562 standard curve was 10,000–0.1 pg/µL (r² ≥ 0.999). The accuracy was examined using NIST SRM 2372a, and its components A, B, and C were quantified with differences of −29.4%, −35.0%, and −22.0%, respectively, against the mtDNA concentrations calculated from published NIST data. We also examined the specificity of the primers, stability of the reaction mix, precision, tolerance against PCR inhibitors, and cross-reactivity against DNA from various animal taxa. Our newly developed mtDNA quantification method is expected to be useful for forensic mtDNA analysis.     

Sequence polymorphisms of mtDNA HV1, HV2, and HV3 regions in the Malay population of Peninsular Malaysia

The uniparentally inherited mitochondrial DNA (mtDNA) is in the limelight for the past two decades, in studies relating to demographic history of mankind and in forensic kinship testing. In this study, human mtDNA hypervariable segments 1, 2, and 3 (HV1, HV2, and HV3) were analyzed in 248 unrelated Malay individuals in Peninsular Malaysia. Combined analyses of HV1, HV2, and HV3 revealed a total of 180 mtDNA haplotypes with 149 unique haplotypes and 31 haplotypes occurring in more than one individual. The genetic diversity was estimated to be 99.47%, and the probability of any two individuals sharing the same mtDNA haplotype was 0.93%. The most frequent mtDNA haplotype (73, 146, 150, 195, 263, 315.1C, 16140, 16182C, 16183C, 16189, 16217, 16274, and 16335) was shared by 11 (4.44%) individuals. The nucleotide diversity and mean of pair-wise differences were found to be 0.036063 ± 0.020101 and 12.544022 ± 6.230486, respectively.     

Sequence polymorphisms of mtDNA HV1, HV2 and HV3 regions in eight population groups living in Taiwan

Analysis of human mitochondrial DNA (mtDNA) polymorphisms in the D-loop region has become a useful tool in forensic casework and matrilineal origin research. In this study, the mtDNA D-loop region including hypervariable region 1 (HV1), hypervariable region 2 (HV2), segment between HV1 and HV2 (7S DNA spanned region), and extended hypervariable region 3 (HV3ex) was sequenced in 539 unrelated individuals from eight population groups living in Taiwan. Combined analyses of the complete D-loop revealed a total of 383 haplotypes with 319 unique haplotypes. The probability of any two individuals sharing the same mtDNA haplotype decreased as the combination of control region segments extended and reached 0.48% with the combination of a complete D-loop region. Sequence variants in HV3ex can further discriminate the haplotypes in some population groups. Phylogenetic haplogroups of these subjects were analyzed. The multidimensional scaling plots of these population groups, constructed based on sequence of the complete D-loop, demonstrated a clear matrilineal genetic substructure in this area. In conclusion, this database of mtDNA complete D-loop sequence including HV3 can serve as a reference for forensic identification. Sequence polymorphisms of the D-loop located outside the HV1 and HV2 may be helpful in further haplogroup characterization.     

Mitochondrial DNA population data of HV1 and HV2 sequences from Japanese individuals

Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were determined for 1204 unrelated Japanese individuals. A total of 741 different mtDNA haplotypes were found, 157 of which were seen in multiple individuals. Twenty-seven of these individuals showed point heteroplasmy. The most frequent haplotype (16223T-16362C-73G-263G-315.1C) was found in 31 individuals and the second most frequent haplotypes (16129A-16223T- 16362C-73G-152C-263G-309.1C-315.1C) was found in 24 individuals. The haplotypes diversity and random match probability were calculated to be 0.9969 and 0.0040, respectively.     

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120–139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data.     

Assessment of the ForenSeq mtDNA control region kit and comparison of orthogonal technologies

The ForenSeq® mtDNA Control Region Kit, MiSeq FGx®, and Universal Analysis Software (UAS) were assessed to better define the performance and limitations of the system with forensically relevant samples to provide data for its transition into practice. A total of six MiSeq FGx sequencing runs of ForenSeq mtDNA Control Region kit, three runs of additional orthogonal sequencing chemistries, and Sanger sequencing results for 14 samples were used to test for concordance. Sensitivity, reproducibility, mixture detection studies, as well as studies to measure the performance of amplification and sequencing controls were performed. The use and reliability of the UAS for data analysis was also examined. With a variety of sample types and controls representing many mitochondrial haplotypes, the recently developed mtDNA Control Region Kit, with the MiSeq FGx and UAS, was found to be fit for purpose as reliable, reproducible, and robust. Sensitivity down to 1 pg of input genomic DNA was demonstrated, which allows the system to offer low limits of detection for better interrogation of potential heteroplasmy in samples. Concerns for implementing next generation sequencing (NGS) for mtDNA in laboratories were addressed in this research, including initial template quantification and confirmation of haplotypes generated by UAS software regarding length-based polymorphisms. To improve performance with forensic samples, laboratories could implement mitochondrial-specific qPCR assays for quantification and perform the optional manual normalization protocol. Additional optimization on sample multiplexing can provide methods that either increase sensitivity or cost efficiency of the assay.     

DNA profile of dog feces as evidence to solve a homicide

Dog fecal samples were collected at the crime scene and from the shoes of the suspect to see whether they could be linked. DNA was genotyped using a 145 bp fragment containing a 60 bp hotspot region of the mitochondrial DNA (mtDNA) control region. Once the species origin was identified, sequences were aligned with the 23 canine haplotypes defined, showing that evidence and reference had 100% identity with haplotype 5. The frequency of haplotype 5 and the exclusion power of the reference population were 0.056 and 0.89, respectively. The forensic index showed that it was 20 times more likely that the evidence belonged to the reference dog than to some other unknown animal. The results support that the mtDNA hypervariable region 1 (HV1) is a good alternative for typing in trace or degraded casework samples when the STR panel fails, and demonstrate the utility of domestic animal samples to give additional information to solve human legal cases.     

Investigation of mtDNA control region sequences in an Egyptian population sample

The sequences of mitochondrial DNA (mtDNA) control region were investigated in 101 unrelated individuals living in the northern region of Nile delta (Gharbia, N = 55 and Kafrelsheikh, N = 46). DNA was extracted from blood stained filter papers or buccal swabs. HV1, HV2 and HV3 were PCR amplified and sequenced; the resulted sequences were aligned and compared with revised Cambridge sequence (rCRS). The results revealed presence of total 93 different haplotypes, 86 of them are unique and 7 are shared haplotypes, the most common haplotype, was observed with a frequency, 2.97% of population sample. High mtDNA diversity was observed with genetic diversity and power of discrimination, 0.9982 and 0.9883, respectively. In this dataset the west Eurasian haplogroups predominated over the African haplogroups. The results would be useful for forensic examinations and human genetic studies.     

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     

Whole mitochondrial genomes assembled from thermally altered forensic bones and teeth

The recovery and analysis of genetic material obtained from thermally altered human bones and teeth are increasingly important to forensic investigations, especially in cases where soft-tissue identification is no longer possible. Although little is known about how these fire-related processes affect DNA degradation over time, next-generation sequencing technology in combination with traditional osteobiographical applications may provide us clues to these questions. In this study, we compare whole mitochondrial genome data generated using two different DNA extraction methods from 27 thermally altered samples obtained from fire victims (Maricopa County, Arizona) . DNA extracts were converted to double-stranded DNA libraries and enriched for whole mitochondrial DNA (mtDNA) using synthetic biotinylated RNA baits, then sequenced on an Illumina MiSeq. We processed the mitochondrial data using an in-house computational pipeline (MitoPipe1.0) composed of ancient DNA and modern genomics applications, then compared the resulting information across the two extraction types and five burn categories. Our analysis shows that DNA fragmentation increases with temperature, but that the acute insult from fire combined with the lack of water is insufficient to produce 5′ and 3′ terminal deamination characteristic of ancient DNA. Our data also suggest an acute and significant point of DNA degradation between 350 °C and 550 °C, and that the likelihood of generating high quality mtDNA haplogroup calls decreases significantly at temperatures > 550 °C. This research is part of a concerted effort to understand how fire affects our ability to generate genetic profiles suitable for forensic identification purposes.     

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.     

Genetic analysis of the presumptive blood from Louis XVI, King of France

A text on a pyrographically decorated gourd dated to 1793 explains that it contains a handkerchief dipped with the blood of Louis XVI, king of France, after his execution. Biochemical analyses confirmed that the material contained within the gourd was blood. The mitochondrial DNA (mtDNA) hypervariable region 1 (HVR1) and 2 (HVR2), the Y-chromosome STR profile, some autosomal STR markers and a SNP in HERC2 gene associated to blue eyes, were retrieved, and some results independently replicated in two different laboratories. The uncommon mtDNA sequence retrieved can be attributed to a N1b haplotype, while the novel Y-chromosome haplotype belongs to haplogroup G2a. The HERC2 gene showed that the subject analyzed was a heterozygote, which is compatible with a blue-eyed person, as king Louis XVI was. To confirm the identity of the subject, an analysis of the dried heart of his son, Louis XVII, could be undertaken.     

An annotated mtDNA database

We have compiled a database of mitochondrial DNA (mtDNA) control region, hypervariable regions 1 (HVR1) and 2 (HVR2) sequences of a total of 14,138 individuals compiled from 103 mtDNA publications before 1 January 2000, 13 data sets published in 2000 and 2001 and 2 unpublished data sets of Iraqi Kurds and Indians from Kerala. By contacting the authors and by other means, we have confirmed and corrected sequence errors, eliminated duplications and harmonised the sequence format. These changes affected all but 26 of the 116 publications. Furthermore, we have implemented a geographic information system (“mtradius”) which searches for closest matches to a given mtDNA control region sequence and displays them on a geographic map. A potential application is to estimate a chance matching probability when a forensic stain and a suspect have an identical mtDNA sequence: we suggest that the geographic area with the highest frequency of closely related mtDNA sequence types may be used to define a reference population to give the suspect the maximum benefit of doubt in accordance with the ceiling principle. Received: 14 August 2000 / Accepted: 22 December 2000     

Evaluation of the MHSeqTyper47 kit for forensically challenging DNA samples

Microhaplotypes have been highly regarded for forensic mixture DNA deconvolution because they do not experience interference from stutters in the same way as short tandem repeat markers, and they tend to be more polymorphic than single nucleotide polymorphism markers. However, forensic microhaplotype kits have not been reported. The MHSeqTyper47 kit genotypes 47 microhaplotype loci. In this study, MiSeq FGx sequencing metrics for MHSeqTyper47 were presented, and the genotyping accuracy of this kit was examined. The sensitivity of MHSeqTyper47 reached 62.5 pg, and full genotyping results were obtained from degraded DNA samples with degradation indexes ≤ 3.00. Full genotypes were obtained in the presence of 100 ng/μL tannin, 50 μM heme, 25 ng/μL humic acid, and 1.25 μg/μL indigo dye. In DNA mixture studies, a minimum of 31 loci of the minor contributor were correctly genotyped at 1:99 or 99:1 mixing ratios, with the cumulative random matching probability of these loci reaching 4.54 × 10⁻²⁵. Mixing ratios could be reliably predicted from two-donor DNA mixtures based on the loci with four called alleles. Taken together, these data showed that the MHSeqTyper47 kit was effective for forensically challenging DNA analysis.     

A rapid mtDNA assay of 22 SNPs in one multiplex reaction increases the power of forensic testing in European Caucasians

We have developed a multiplex mitochondrial (mtDNA) assay of 21 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable region 1 (HVR1) and hypervariable region 2 (HVR2) that can be amplified in a single reverse touchdown polymerase chain reaction. Single base extension using the SNaPshot technique is also carried out as one multiplex. Besides the nine major European haplogroups (i.e. H, I, J, K, T, U, V, W, and X), 16 additional subclades (i.e. N1, X2, X2b, U2′-4/7′-9′, J/T, J1, J1c, HV, H1, H1a1, H1c, H3, H4, H6a, H7a H10) can be detected and classified into a phylogenetic mtDNA tree. By analyzing 130 Caucasoid samples from Germany, 36 different haplotypes were found resulting in a power of discrimination of 93.2%. Although 49% of all samples belonged to superhaplogroup H, the most common haplotype, i.e., haplogroup-specific SNPs plus haplogroup unspecific SNPs, had a frequency of only 18%. This assay is applicable for high-throughput mtDNA analysis and forensic mass screening. It will give additional information to the common control region sequencing of HVR1 and HVR2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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     

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     

DNA typing from human faeces

A method has been developed for the forensic analysis of faeces by DNA amplification and direct sequencing of a polymorphic segment of mitochondrial DNA. Starting from as little as 10 mg wet weight of faeces, DNA was extracted by a variety of protocols and amplified using primers specific to hypervariable region I of the mitochondrial control region. The resulting amplification products were sequenced in solid phase using an automated DNA sequencer. In total, mtDNA sequences were generated from the faeces of nine Caucasians and compared with sequences generated from their respective blood samples. Sequences of faeces and blood samples from the same individual were identical in every case, but a range of 1–10 nucleotide differences was observed between individuals, with an average sequence variation of approximately 4.88 per 400 bp. Of the various extraction protocols assessed in this study, greatest success rates were achieved using magnetisable beads to bind and purify the DNA. STR analysis of DNA extracted from faeces was not routinely possible.     

A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples

The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups.