A statistical framework for the interpretation of mtDNA mixtures: forensic and medical applications

Background

Mitochondrial DNA (mtDNA) variation is commonly analyzed in a wide range of different biomedical applications. Cases where more than one individual contribute to a stain genotyped from some biological material give rise to a mixture. Most forensic mixture cases are analyzed using autosomal markers. In rape cases, Y-chromosome markers typically add useful information. However, there are important cases where autosomal and Y-chromosome markers fail to provide useful profiles. In some instances, usually involving small amounts or degraded DNA, mtDNA may be the only useful genetic evidence available. Mitochondrial DNA mixtures also arise in studies dealing with the role of mtDNA variation in tumorigenesis. Such mixtures may be generated by the tumor, but they could also originate in vitro due to inadvertent contamination or a sample mix-up.

Methods/Principal Findings

We present the statistical methods needed for mixture interpretation and emphasize the modifications required for the more well-known methods based on conventional markers to generalize to mtDNA mixtures. Two scenarios are considered. Firstly, only categorical mtDNA data is assumed available, that is, the variants contributing to the mixture. Secondly, quantitative data (peak heights or areas) on the allelic variants are also accessible. In cases where quantitative information is available in addition to allele designation, it is possible to extract more precise information by using regression models. More precisely, using quantitative information may lead to a unique solution in cases where the qualitative approach points to several possibilities. Importantly, these methods also apply to clinical cases where contamination is a potential alternative explanation for the data.

Conclusions/Significance

We argue that clinical and forensic scientists should give greater consideration to mtDNA for mixture interpretation. The results and examples show that the analysis of mtDNA mixtures contributes substantially to forensic casework and may also clarify erroneous claims made in clinical genetics regarding tumorigenesis.     

Raw data graphing: an informative but under‐utilized tool for the analysis of multivariate abundances

Abstract In exploring the relationship between multivariate abundance data and environmental variables, a rarely used approach is to graph raw data separately for each different taxon. It is proposed that such raw data graphs become part of the standard toolset for graphing and analysing multivariate abundances. The key advantage of this approach is that axis scales have quantitative interpretations, enabling quantitative interpretation of patterns in abundance. In contrast, ordinations only present qualitative information. Ordinations are useful for inferring overall, qualitative patterns and raw data graphing is a complementary tool of greater use for answering more specific questions, aimed at a deeper understanding the ecology of a community. It is demonstrated using some well‐known examples that our understanding of the nature of associations can be considerably improved by using raw data graphs, even when only plotting a subset of variables. One example describes how an often‐cited dataset has been misinterpreted in key methodological papers, because data were interpreted from ordinations alone, with no consideration of plots of the raw data.     

Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection

DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methylation. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16^INK4a) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP.     

Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons

Genetically modified (GM) cotton lines have been approved for commercialization and widely cultivated in many countries, especially in China. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM cottons, we report here the validation of the cotton (Gossypium hirsutum) endogenous reference control gene, Sad1, using conventional and real-time (RT)-PCR methods. Both methods were tested on 15 different G. hirsutum cultivars, and identical amplicons were obtained with all of them. No amplicons were observed when DNA samples from three species of genus Gossypium, Arabidopsis thaliana, maize, and soybean and others were used as amplified templates, demonstrating that these two systems are specific for the identification and quantification of G. hirsutum. The results of Southern blot analysis also showed that the Sad1 gene was two copies in these 15 different G. hirsutum cultivars. Furthermore, one multiplex RT-quantitative PCR employing this gene as an endogenous reference gene was designed to quantify the Cry1A(c) gene modified from Bacillus thuringiensis (Bt) in the insect-resistant cottons, such as Mon531 and GK19. The quantification detection limit of the Cry1A(c) and Sad1 genes was as low as 10 pg of genomic DNA. These results indicat that the Sad1 gene can be used as an endogenous reference gene for both qualitative and quantitative PCR detection of GM cottons.     

Quantitative high-pressure liquid chromatographic analysis of methylated purines in DNA of rats treated with chemical carcinogens

A rapid, sensitive method for the quantitative measurement of certain major and modified purines in DNA of carcinogen-treated animals is presented. DNA hydrolysates are analyzed by high-pressure liquid chromatography combined with fluorescence detection and electronic integration of peaks. Limits of detection are approximately 7 ng for 7-methylguanine and 150 pg for O⁶-methylguanine. Between 100 and 250 μg target organ DNA from animals treated with several carcinogens was shown to contain readily detectable amounts of these methylated bases. The method provides results comparable to those obtained with conventional methods using radioactively labeled carcinogens.     

Quantitative intra-short interspersed element PCR for species-specific DNA identification

We have designed and evaluated four assays based upon PCR amplification of short interspersed elements (SINEs) for species-specific detection and quantitation of bovine, porcine, chicken, and ruminant DNA. The need for these types of approaches has increased drastically in response to the bovine spongiform encephalopathy epidemic. Using SYBR Green-based detection, the minimum effective quantitation levels were 0.1, 0.01, 5, and 1 pg of starting DNA template using our bovine, porcine, chicken, and ruminant species-specific SINE-based PCR assays, respectively. Background cross-amplification with DNA templates derived from 14 other species was negligible. Species specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect trace quantities of species-specific DNA from mixed (complex) sources. Bovine DNA was detected at 0.005% (0.5 pg), porcine DNA was detected at 0.0005% (0.05 pg), and chicken DNA was detected at 0.05% (5 pg) in a 10-ng mixture of bovine, porcine, and chicken DNA templates. We also tested six commercially purchased meat products using these assays. The SINE-based PCR methods we report here are species-specific, are highly sensitive, and will improve the detection limits for DNA sequences derived from these species.     

Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method

A method employing loop-mediated isothermal amplification (LAMP) of 18S ribosomal RNA gene was developed to detect Acanthamoeba in contact lens cases. A prevalence of 7% (10/150) was detected, with 100% sensitivity and 100% specificity when compared with the standard culture technique. Using visual inspection of turbidity a minimum of 10 pg of Acanthamoeba DNA could be detected, 10 times more sensitive than quantitative PCR employing two of the LAMP primers. The production of LAMP amplicons was confirmed by gel-electrophoresis and ethidium bromide staining. The LAMP procedure takes less than 2 h to perform and will be useful for incorporation into a point-of-care screening of suspected Acanthamoeba infection.     

Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli

We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50–160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.     

Genotypic diversity,molecular markers and spatial distribution of genets in clonal plants,a literature survey

We present a literature survey of studies using molecular markers to investigate genet diversity and structure in clonal plants. The data from 40 studies comprised 45 species of which only two were studied by DNA methods, the rest by isozyme markers. Less than one third of the studies provided information about the spatial distribution of individual genets within populations, and only 12.5% of the studies used mapping of all ramets within plots or part of the population in combination with identification of multilocus genotypes. We also present two case studies. InGlechoma hederacea morphological criteria were used to select clones. Multi-samples of ramets within these “clones” turned out to be variable using isozymes indicating that these “clones” in most cases consisted of several genets. One individual multilocus genotype covered tens of square metres. InHylocomium splendens samples from 10×10 cm plots usually consisted of a mixture of multilocus genotypes, but occasionally the whole plot consisted of one genet.     

Comparing whole‐genome shotgun sequencing and DNA metabarcoding approaches for species identification and quantification of pollen species mixtures

Molecular identification of mixed‐species pollen samples has a range of applications in various fields of research. To date, such molecular identification has primarily been carried out via amplicon sequencing, but whole‐genome shotgun (WGS) sequencing of pollen DNA has potential advantages, including (1) more genetic information per sample and (2) the potential for better quantitative matching. In this study, we tested the performance of WGS sequencing methodology and publicly available reference sequences in identifying species and quantifying their relative abundance in pollen mock communities. Using mock communities previously analyzed with DNA metabarcoding, we sequenced approximately 200Mbp for each sample using Illumina HiSeq and MiSeq. Taxonomic identifications were based on the Kraken k‐mer identification method with reference libraries constructed from full‐genome and short read archive data from the NCBI database. We found WGS to be a reliable method for taxonomic identification of pollen with near 100% identification of species in mixtures but generating higher rates of false positives (reads not identified to the correct taxon at the required taxonomic level) relative to rbcL and ITS2 amplicon sequencing. For quantification of relative species abundance, WGS data provided a stronger correlation between pollen grain proportion and sequence read proportion, but diverged more from a 1:1 relationship, likely due to the higher rate of false positives. Currently, a limitation of WGS‐based pollen identification is the lack of representation of plant diversity in publicly available genome databases. As databases improve and costs drop, we expect that eventually genomics methods will become the methods of choice for species identification and quantification of mixed‐species pollen samples.     

Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA.

The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. DNA strand cleavage via beta-elimination (beta-lyase) activity coupled with MutY's removal of misincorporated adenine bases was sought using both qualitative and quantitative methods. The qualitative assays demonstrate formation of a Schiff base intermediate which is characteristic of DNA glycosylases catalyzing a concomitant beta-lyase reaction. Borohydride reduction of the Schiff base results in the formation of a covalent DNA-MutY adduct which is easily detected in SDS-PAGE experiments. However, quantitative activity assays which monitor DNA strand scission accompanying base release suggest MutY behaves as a simple monofunctional glycosylase. Treatment with base effects DNA strand cleavage at apurinic/apyrimidinic (AP) sites arising via simple glycosylase activity. The amount of cleaved DNA in MutY reactions treated with base is much greater than that in non-base treated reactions, indicating that AP site generation by MutY is not associated with a concomitant beta-lyase step. As standards, identical assays were performed with a known monofunctional enzyme (uracil DNA glycosylase) and a known bifunctional glycosylase/lyase (FPG), the results of which were used in comparison with those of the MutY experiments. The apparent inconsistency between the data obtained for MutY by the qualitative and quantitative methods underscores the current debate surrounding the catalytic activity of this enzyme, and a detailed explanation of this controversy is proposed. The work presented here lays ground for the identification of specific active site residues responsible for the chemical mechanism of MutY enzyme catalysis.     

Quantitation of Microorganisms in Sputum

A method of quantitating microbial cultures of homogenized sputum has been devised. Possible application of this method to the problem of determining the etiologic agent of lower-respiratory-tract infections has been studied to determine its usefulness as a guide in the management of these infections. Specimens were liquefied by using an equal volume of 2% N-acetyl-L-cysteine. The liquefied sputum suspension was serially diluted to 10^-1, 10^-3, 10^-5, and 10^-7. These dilutions were plated on appropriate media by using an 0.01-ml calibrated loop; they were incubated and examined by standard diagnostic methods. Quantitation of fresh sputum from patients with pneumonia prior to antimicrobial therapy revealed that probable pathogens were present in populations of 10⁷ organisms/ml or greater. Normal oropharyngeal flora did not occur in these numbers before therapy. Comparison of microbial counts on fresh and aged sputum showed that it is necessary to use only fresh specimens, since multiplication or death alters both quantitative and qualitative findings. Proper collection and quantitative culturing of homogenized sputum provided information more directly applicable to patient management than did qualitative routine methods. Not only was the recognition of the probable pathogenic organism in pneumonia patients improved, but serial quantitative cultures were particularly useful in recognizing the emergence of superinfections and in evaluating the efficacy of antimicrobial therapy.     

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.     

NotI passporting to identify species composition of complex microbial systems

We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.     

Assay for the quantification of intact/fragmented genomic DNA

This study shows that the accuracy of the quantification of genomic DNA by the commonly used Hoechst- and PicoGreen-based assays is drastically affected by its degree of fragmentation. Specifically, it was shown that these assays underestimate by 70% the concentration of double-stranded DNA (dsDNA) with sizes less than 23 kb. On the other hand, DNA sizes greater and less than approximately 23 kb are commonly characterized as intact and fragmented genomic DNA, respectively, by the agarose electrophoresis DNA smearing assay and are evaluated only qualitatively by this assay. The need for accurate quantification of fragmented and total genomic DNA, combined with the lack of specific, reliable, and simple quantitative methods, prompted us to develop a Hoechst/PicoGreen-based fluorescent assay that quantifies both types of DNA. This assay addresses these problems, and in its Hoechst and PicoGreen version it accurately quantifies dsDNA as being either intact (>or=23 kb) or fragmented (<23 kb) in concentrations as low as 3 ng ml-1 or 5 pg ml-1 with Hoechst or PicoGreen, respectively, as well as the individual fractions of intact/fragmented DNA existing in any proportions in a total DNA sample in concentrations as low as 10 ng ml-1 or 15 pg ml-1 with Hoechst or PicoGreen, respectively. Because the assay discriminates total genomic DNA in the two size ranges (>or=23 and <23 kb) and quantitates them, it is proposed as the quantitative replacement of the agarose gel electrophoresis genomic DNA smearing assay.     

Identifying insects with incomplete DNA barcode libraries, African fruit flies (Diptera: Tephritidae) as a test case

We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods.