Genes

Research

47 pages, 2531 KiB

Open AccessArticle

Human Mitochondrial Control Region and mtGenome: Design and Forensic Validation of NGS Multiplexes, Sequencing and Analytical Software

by Cydne L. Holt, Kathryn M. Stephens, Paulina Walichiewicz, Keenan D. Fleming, Elmira Forouzmand and Shan-Fu Wu

Genes 2021, 12(4), 599; https://doi.org/10.3390/genes12040599 - 19 Apr 2021

Cited by 25 | Viewed by 7113

Abstract

Forensic mitochondrial DNA (mtDNA) analysis conducted using next-generation sequencing (NGS), also known as massively parallel sequencing (MPS), as compared to Sanger-type sequencing brings modern advantages, such as deep coverage per base (herein referred to as read depth per base pair (bp)), simultaneous sequencing [...] Read more.

Forensic mitochondrial DNA (mtDNA) analysis conducted using next-generation sequencing (NGS), also known as massively parallel sequencing (MPS), as compared to Sanger-type sequencing brings modern advantages, such as deep coverage per base (herein referred to as read depth per base pair (bp)), simultaneous sequencing of multiple samples (libraries) and increased operational efficiencies. This report describes the design and developmental validation, according to forensic quality assurance standards, of end-to-end workflows for two multiplexes, comprised of ForenSeq mtDNA control region and mtDNA whole-genome kits the MiSeq FGx^TM instrument and ForenSeq universal analysis software (UAS) 2.0/2.1. Polymerase chain reaction (PCR) enrichment and a tiled amplicon approach target small, overlapping amplicons (60–150 bp and 60–209 bp for the control region and mtGenome, respectively). The system provides convenient access to data files that can be used outside of the UAS if desired. Studies assessed a range of environmental and situational variables, including but not limited to buccal samples, rootless hairs, dental and skeletal remains, concordance of control region typing between the two multiplexes and as compared to orthogonal data, assorted sensitivity studies, two-person DNA mixtures and PCR-based performance testing. Limitations of the system and implementation considerations are discussed. Data indicated that the two mtDNA multiplexes, MiSeq FGx and ForenSeq software, meet or exceed forensic DNA quality assurance (QA) guidelines with robust, reproducible performance on samples of various quantities and qualities. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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16 pages, 2872 KiB

Open AccessArticle

Graph Algorithms for Mixture Interpretation

by Benjamin Crysup, August E. Woerner, Jonathan L. King and Bruce Budowle

Genes 2021, 12(2), 185; https://doi.org/10.3390/genes12020185 - 27 Jan 2021

Cited by 3 | Viewed by 2574

Abstract

The scale of genetic methods are presently being expanded: forensic genetic assays previously were limited to tens of loci, but now technologies allow for a transition to forensic genomic approaches that assess thousands to millions of loci. However, there are subtle distinctions between [...] Read more.

The scale of genetic methods are presently being expanded: forensic genetic assays previously were limited to tens of loci, but now technologies allow for a transition to forensic genomic approaches that assess thousands to millions of loci. However, there are subtle distinctions between genetic assays and their genomic counterparts (especially in the context of forensics). For instance, forensic genetic approaches tend to describe a locus as a haplotype, be it a microhaplotype or a short tandem repeat with its accompanying flanking information. In contrast, genomic assays tend to provide not haplotypes but sequence variants or differences, variants which in turn describe how the alleles apparently differ from the reference sequence. By the given construction, mitochondrial genetic assays can be thought of as genomic as they often describe genetic differences in a similar way. The mitochondrial genetics literature makes clear that sequence differences, unlike the haplotypes they encode, are not comparable to each other. Different alignment algorithms and different variant calling conventions may cause the same haplotype to be encoded in multiple ways. This ambiguity can affect evidence and reference profile comparisons as well as how “match” statistics are computed. In this study, a graph algorithm is described (and implemented in the MMDIT (Mitochondrial Mixture Database and Interpretation Tool) R package) that permits the assessment of forensic match statistics on mitochondrial DNA mixtures in a way that is invariant to both the variant calling conventions followed and the alignment parameters considered. The algorithm described, given a few modest constraints, can be used to compute the “random man not excluded” statistic or the likelihood ratio. The performance of the approach is assessed in in silico mitochondrial DNA mixtures. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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15 pages, 2261 KiB

Open AccessArticle

Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

by Catarina Xavier, Mayra Eduardoff, Barbara Bertoglio, Christina Amory, Cordula Berger, Andrea Casas-Vargas, Johannes Pallua and Walther Parson

Genes 2021, 12(2), 146; https://doi.org/10.3390/genes12020146 - 22 Jan 2021

Cited by 36 | Viewed by 12982

Abstract

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in [...] Read more.

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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21 pages, 3585 KiB

Open AccessArticle

A Continuous Statistical Phasing Framework for the Analysis of Forensic Mitochondrial DNA Mixtures

by Utpal Smart, Jennifer Churchill Cihlar, Sammed N. Mandape, Melissa Muenzler, Jonathan L. King, Bruce Budowle and August E. Woerner

Genes 2021, 12(2), 128; https://doi.org/10.3390/genes12020128 - 20 Jan 2021

Cited by 9 | Viewed by 4347

Abstract

Despite the benefits of quantitative data generated by massively parallel sequencing, resolving mitotypes from mixtures occurring in certain ratios remains challenging. In this study, a bioinformatic mixture deconvolution method centered on population-based phasing was developed and validated. The method was first tested on [...] Read more.

Despite the benefits of quantitative data generated by massively parallel sequencing, resolving mitotypes from mixtures occurring in certain ratios remains challenging. In this study, a bioinformatic mixture deconvolution method centered on population-based phasing was developed and validated. The method was first tested on 270 in silico two-person mixtures varying in mixture proportions. An assortment of external reference panels containing information on haplotypic variation (from similar and different haplogroups) was leveraged to assess the effect of panel composition on phasing accuracy. Building on these simulations, mitochondrial genomes from the Human Mitochondrial DataBase were sourced to populate the panels and key parameter values were identified by deconvolving an additional 7290 in silico two-person mixtures. Finally, employing an optimized reference panel and phasing parameters, the approach was validated with in vitro two-person mixtures with differing proportions. Deconvolution was most accurate when the haplotypes in the mixture were similar to haplotypes present in the reference panel and when the mixture ratios were neither highly imbalanced nor subequal (e.g., 4:1). Overall, errors in haplotype estimation were largely bounded by the accuracy of the mixture’s genotype results. The proposed framework is the first available approach that automates the reconstruction of complete individual mitotypes from mixtures, even in ratios that have traditionally been considered problematic. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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12 pages, 1651 KiB

Open AccessArticle

Reducing the Number of Mismatches between Hairs and Buccal References When Analysing mtDNA Heteroplasmic Variation by Massively Parallel Sequencing

by Kristiaan J. van der Gaag, Stijn Desmyter, Sophie Smit, Lourdes Prieto and Titia Sijen

Genes 2020, 11(11), 1355; https://doi.org/10.3390/genes11111355 - 16 Nov 2020

Cited by 17 | Viewed by 2863

Abstract

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold [...] Read more.

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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Graphical abstract

31 pages, 4291 KiB

Open AccessArticle

Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel

by Jennifer Churchill Cihlar, Christina Amory, Robert Lagacé, Chantal Roth, Walther Parson and Bruce Budowle

Genes 2020, 11(11), 1345; https://doi.org/10.3390/genes11111345 - 13 Nov 2020

Cited by 34 | Viewed by 4825

Abstract

For the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology—Precision ID mtDNA [...] Read more.

For the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology—Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge—has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra- and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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17 pages, 2077 KiB

Open AccessArticle

Mitochondrial Sequencing of Missing Persons DNA Casework by Implementing Thermo Fisher’s Precision ID mtDNA Whole Genome Assay

by Daniela Cuenca, Jessica Battaglia, Michelle Halsing and Sandra Sheehan

Genes 2020, 11(11), 1303; https://doi.org/10.3390/genes11111303 - 4 Nov 2020

Cited by 27 | Viewed by 4180

Abstract

The advent of massively parallel sequencing (MPS) in the past decade has opened the doors to mitochondrial whole-genome sequencing. Mitochondrial (mt) DNA is used in forensics due to its high copy number per cell and maternal mode of inheritance. Consequently, we have implemented [...] Read more.

The advent of massively parallel sequencing (MPS) in the past decade has opened the doors to mitochondrial whole-genome sequencing. Mitochondrial (mt) DNA is used in forensics due to its high copy number per cell and maternal mode of inheritance. Consequently, we have implemented the Thermo Fisher Precision ID mtDNA Whole Genome panel coupled with the Ion Chef™ and Ion S5™ for MPS analysis in the California Department of Justice, Missing Persons DNA Program. Thirty-one mostly challenging samples (degraded, inhibited, low template, or mixed) were evaluated for this study. The majority of these samples generated single source full or partial genome sequences with MPS, providing information in cases where previously there was none. The quantitative and sensitive nature of MPS analysis was beneficial, but also led to detection of low-level contaminants. In addition, we found Precision ID to be more susceptible to inhibition than our legacy Sanger assay. Overall, the success rate (full single source hypervariable regions I and II (HVI/HVII) for Sanger and control region for MPS result) for these challenging samples increased from 32.3% with Sanger sequencing to 74.2% with the Precision ID assay. Considering the increase in success rate, the simple workflow and the higher discriminating potential of whole genome data, the Precision ID platform is a significant improvement for the CA Department of Justice Missing Persons DNA Program. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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24 pages, 1517 KiB

Open AccessArticle

Platinum-Quality Mitogenome Haplotypes from United States Populations

by Cassandra R. Taylor, Kevin M. Kiesler, Kimberly Sturk-Andreaggi, Joseph D. Ring, Walther Parson, Moses Schanfield, Peter M. Vallone and Charla Marshall

Genes 2020, 11(11), 1290; https://doi.org/10.3390/genes11111290 - 29 Oct 2020

Cited by 19 | Viewed by 6004

Abstract

A total of 1327 platinum-quality mitochondrial DNA haplotypes from United States (U.S.) populations were generated using a robust, semi-automated next-generation sequencing (NGS) workflow with rigorous quality control (QC). The laboratory workflow involved long-range PCR to minimize the co-amplification of nuclear mitochondrial DNA segments [...] Read more.

A total of 1327 platinum-quality mitochondrial DNA haplotypes from United States (U.S.) populations were generated using a robust, semi-automated next-generation sequencing (NGS) workflow with rigorous quality control (QC). The laboratory workflow involved long-range PCR to minimize the co-amplification of nuclear mitochondrial DNA segments (NUMTs), PCR-free library preparation to reduce amplification bias, and high-coverage Illumina MiSeq sequencing to produce an average per-sample read depth of 1000 × for low-frequency (5%) variant detection. Point heteroplasmies below 10% frequency were confirmed through replicate amplification, and length heteroplasmy was quantitatively assessed using a custom read count analysis tool. Data analysis involved a redundant, dual-analyst review to minimize errors in haplotype reporting with additional QC checks performed by EMPOP. Applying these methods, eight sample sets were processed from five U.S. metapopulations (African American, Caucasian, Hispanic, Asian American, and Native American) corresponding to self-reported identity at the time of sample collection. Population analyses (e.g., haplotype frequencies, random match probabilities, and genetic distance estimates) were performed to evaluate the eight datasets, with over 95% of haplotypes unique per dataset. The platinum-quality mitogenome haplotypes presented in this study will enable forensic statistical calculations and thereby support the usage of mitogenome sequencing in forensic laboratories. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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10 pages, 1011 KiB

Open AccessArticle

Pathogenic Variant Filtering for Mitochondrial Genome Haplotype Reporting

by Charla Marshall, Kimberly Sturk-Andreaggi, Joseph D. Ring, Arne Dür and Walther Parson

Genes 2020, 11(10), 1140; https://doi.org/10.3390/genes11101140 - 28 Sep 2020

Cited by 6 | Viewed by 3171

Abstract

Given the enhanced discriminatory power of the mitochondrial DNA (mtDNA) genome (mitogenome) over the commonly sequenced control region (CR) portion, the scientific merit of mitogenome sequencing is generally accepted. However, many laboratories remain beholden to CR sequencing due to privacy policies and legal [...] Read more.

Given the enhanced discriminatory power of the mitochondrial DNA (mtDNA) genome (mitogenome) over the commonly sequenced control region (CR) portion, the scientific merit of mitogenome sequencing is generally accepted. However, many laboratories remain beholden to CR sequencing due to privacy policies and legal requirements restricting the use of disease information or coding region (codR) information. In this report, we present an approach to obviate the reporting of sensitive codR data in forensic haplotypes. We consulted the MitoMap database to identify 92 mtDNA codR variants with confirmed pathogenicity. We determined the frequencies of these pathogenic variants in literature-quality and forensic-quality databases to be very low, at 1.2% and 0.36%, respectively. The observed effect of pathogenic variant filtering on random match statistics in 2488 forensic-quality mitogenome haplotypes from four populations was nil. We propose that pathogenic variant filtering should be incorporated into variant calling algorithms for mitogenome haplotype reporting to maximize the discriminatory power of the locus while minimizing the reveal of sensitive genetic information. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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13 pages, 6776 KiB

Open AccessFeature PaperArticle

A Forensic Genomics Approach for the Identification of Sister Marija Crucifiksa Kozulić

by Charla Marshall, Kimberly Sturk-Andreaggi, Erin M. Gorden, Jennifer Daniels-Higginbotham, Sidney Gaston Sanchez, Željana Bašić, Ivana Kružić, Šimun Anđelinović, Alan Bosnar, Miran Čoklo, Anja Petaros, Timothy P. McMahon, Dragan Primorac and Mitchell M. Holland

Genes 2020, 11(8), 938; https://doi.org/10.3390/genes11080938 - 14 Aug 2020

Cited by 7 | Viewed by 4658

Abstract

Sister Marija Krucifiksa Kozulić (1852–1922) was a Croatian nun who is in consideration for beatification by the Vatican, which is facilitated by the identification of her 20th-century remains. Sister Marija was buried in a tomb in Rijeka, Croatia, along with other nuns including [...] Read more.

Sister Marija Krucifiksa Kozulić (1852–1922) was a Croatian nun who is in consideration for beatification by the Vatican, which is facilitated by the identification of her 20th-century remains. Sister Marija was buried in a tomb in Rijeka, Croatia, along with other nuns including her biological sister, Tereza Kozulić (1861–1933). When the remains were exhumed in 2011, they were found in a deteriorated state and commingled with several other sets of remains. Thus, mitochondrial genome sequencing of the long bones was performed to sort the remains by mitochondrial haplotype. Two similar but unique haplotypes belonging to haplogroup H1bu were identified, and samples from these bones were subjected to autosomal short tandem repeat (STR) and single nucleotide polymorphism (SNP) sequencing. Although only partial profiles were obtained, the data were sufficient for kinship analysis with the profile of a paternal niece of Sister Marija (Fides Kozulić). The data indicate that it is 574,195-fold more likely that the two sets of skeletal remains represent 2nd-degree relatives of Fides than sisters who are unrelated to Fides. Although it is impossible to discern which set of remains belongs to Marija and which belongs to Tereza, forensic genomics methods have enabled identification of the sisters. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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11 pages, 1815 KiB

Open AccessArticle

Heteroplasmy and Copy Number in the Common m.3243A>G Mutation—A Post-Mortem Genotype–Phenotype Analysis

by Leila Motlagh Scholle, Stephan Zierz, Christian Mawrin, Claudia Wickenhauser and Diana Lehmann Urban

Genes 2020, 11(2), 212; https://doi.org/10.3390/genes11020212 - 18 Feb 2020

Cited by 33 | Viewed by 4480

Abstract

Different mitochondrial DNA (mtDNA) mutations have been identified to cause mitochondrial encephalopathy, lactate acidosis and stroke-like episodes (MELAS). The underlying genetic cause leading to an enormous clinical heterogeneity associated with m.3243A>G-related mitochondrial diseases is still poorly understood. Genotype–phenotype correlation (heteroplasmy levels and clinical [...] Read more.

Different mitochondrial DNA (mtDNA) mutations have been identified to cause mitochondrial encephalopathy, lactate acidosis and stroke-like episodes (MELAS). The underlying genetic cause leading to an enormous clinical heterogeneity associated with m.3243A>G-related mitochondrial diseases is still poorly understood. Genotype–phenotype correlation (heteroplasmy levels and clinical symptoms) was analysed in 16 patients (15 literature cases and one unreported case) harbouring the m.3243A>G mutation. mtDNA copy numbers were correlated to heteroplasmy levels in 30 different post-mortem tissue samples, including 14 brain samples of a 46-year-old female. In the central nervous system, higher levels of heteroplasmy correlated significantly with lower mtDNA copy numbers. Skeletal muscle levels of heteroplasmy correlated significantly with kidney and liver. There was no significant difference of heteroplasmy levels between clinically affected and unaffected patients. In the patient presented, we found >75% heteroplasmy levels in all central nervous system samples, without harbouring a MELAS phenotype. This underlines previous suggestions, that really high levels in tissues do not automatically lead to a specific phenotype. Missing significant differences of heteroplasmy levels between clinically affected and unaffected patients underline recent suggestions that there are additional factors such as mtDNA copy number and nuclear factors that may also influence disease severity. Full article

(This article belongs to the Special Issue Forensic Mitochondrial Genomics)

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