Showing papers in "Investigative Genetics in 2013"

DNA fingerprinting in forensics: past, present, future.

Abstract: DNA fingerprinting, one of the great discoveries of the late 20th century, has revolutionized forensic investigations. This review briefly recapitulates 30 years of progress in forensic DNA analysis which helps to convict criminals, exonerate the wrongly accused, and identify victims of crime, disasters, and war. Current standard methods based on short tandem repeats (STRs) as well as lineage markers (Y chromosome, mitochondrial DNA) are covered and applications are illustrated by casework examples. Benefits and risks of expanding forensic DNA databases are discussed and we ask what the future holds for forensic DNA fingerprinting.

Inference of human continental origin and admixture proportions using a highly discriminative ancestry informative 41-SNP panel

Abstract: Accurate determination of genetic ancestry is of high interest for many areas such as biomedical research, personal genomics and forensics. It remains an important topic in genetic association studies, as it has been shown that population stratification, if not appropriately considered, can lead to false-positive and -negative results. While large association studies typically extract ancestry information from available genome-wide SNP genotypes, many important clinical data sets on rare phenotypes and historical collections assembled before the GWAS area are in need of a feasible method (i.e., ease of genotyping, small number of markers) to infer the geographic origin and potential admixture of the study subjects. Here we report on the development, application and limitations of a small, multiplexable ancestry informative marker (AIM) panel of SNPs (or AISNP) developed specifically for this purpose. Based on worldwide populations from the HGDP, a 41-AIM AISNP panel for multiplex application with the ABI SNPlex and a subset with 31 AIMs for the Sequenome iPLEX system were selected and found to be highly informative for inferring ancestry among the seven continental regions Africa, the Middle East, Europe, Central/South Asia, East Asia, the Americas and Oceania. The panel was found to be least informative for Eurasian populations, and additional AIMs for a higher resolution are suggested. A large reference set including over 4,000 subjects collected from 120 global populations was assembled to facilitate accurate ancestry determination. We show practical applications of this AIM panel, discuss its limitations for admixed individuals and suggest ways to incorporate ancestry information into genetic association studies. We demonstrated the utility of a small AISNP panel specifically developed to discern global ancestry. We believe that it will find wide application because of its feasibility and potential for a wide range of applications.

DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

Abstract: Background Mitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome.

Bona fide colour: DNA prediction of human eye and hair colour from ancient and contemporary skeletal remains

Abstract: Background: DNA analysis of ancient skeletal remains is invaluable in evolutionary biology for exploring the history of species, including humans. Contemporary human bones and teeth, however, are relevant in forensic DNA analyses that deal with the identification of perpetrators, missing persons, disaster victims or family relationships. They may also provide useful information towards unravelling controversies that surround famous historical individuals. Retrieving information about a deceased person’s externally visible characteristics can be informative in both types of DNA analyses. Recently, we demonstrated that human eye and hair colour can be reliably predicted from DNA using the HIrisPlex system. Here we test the feasibility of the novel HIrisPlex system at establishing eye and hair colour of deceased individuals from skeletal remains of various post-mortem time ranges and storage conditions. Methods: Twenty-one teeth between 1 and approximately 800 years of age and 5 contemporary bones were subjected to DNA extraction using standard organic protocol followed by analysis using the HIrisPlex system. Results: Twenty-three out of 26 bone DNA extracts yielded the full 24 SNP HIrisPlex profile, therefore successfully allowing model-based eye and hair colour prediction. HIrisPlex analysis of a tooth from the Polish general Wladyslaw Sikorski (1881 to 1943) revealed blue eye colour and blond hair colour, which was positively verified from reliable documentation. The partial profiles collected in the remaining three cases (two contemporary samples and a 14th century sample) were sufficient for eye colour prediction. Conclusions: Overall, we demonstrate that the HIrisPlex system is suitable, sufficiently sensitive and robust to successfully predict eye and hair colour from ancient and contemporary skeletal remains. Our findings, therefore, highlight the HIrisPlex system as a promising tool in future routine forensic casework involving skeletal remains, including ancient DNA studies, for the prediction of eye and hair colour of deceased individuals.

Inferring human history in East Asia from Y chromosomes

Abstract: East Asia harbors substantial genetic, physical, cultural and linguistic diversity, but the detailed structures and interrelationships of those aspects remain enigmatic This question has begun to be addressed by a rapid accumulation of molecular anthropological studies of the populations in and around East Asia, especially by Y chromosome studies The current Y chromosome evidence suggests multiple early migrations of modern humans from Africa via Southeast Asia to East Asia After the initial settlements, the northward migrations during the Paleolithic Age shaped the genetic structure in East Asia Subsequently, recent admixtures between Central Asian immigrants and northern East Asians enlarged the genetic divergence between southern and northern East Asia populations Cultural practices, such as languages, agriculture, military affairs and social prestige, also have impacts on the genetic patterns in East Asia Furthermore, application of Y chromosome analyses in the family genealogy studies offers successful showcases of the utility of genetics in studying the ancient history

Response to Grisedale and Van Daal: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method

Abstract: In a recent contribution to this journal Grisedale and Van Daal concluded that a single STR analysis of all available template DNA is to be preferred over replicate analyses and a consensus approach when analyzing low template DNA samples. A single STR analysis approach does not allow for an assessment of the validity of the resulting DNA profile. We argue that the use of replicate amplifications is the best way to objectively quantify the extent of the stochastic variation in the data. By applying consensus methodology and/or a probabilistic model, the interpretation of the data will therefore be more objective and reliable.

Fully integrated, fully automated generation of short tandem repeat profiles

Abstract: Background The generation of short tandem repeat profiles, also referred to as ‘DNA typing,’ is not currently performed outside the laboratory because the process requires highly skilled technical operators and a controlled laboratory environment and infrastructure with several specialized instruments. The goal of this work was to develop a fully integrated system for the automated generation of short tandem repeat profiles from buccal swab samples, to improve forensic laboratory process flow as well as to enable short tandem repeat profile generation to be performed in police stations and in field-forward military, intelligence, and homeland security settings.

Where is the game? Wild meat products authentication in South Africa: a case study

Abstract: Wild animals’ meat is extensively consumed in South Africa, being obtained either from ranching, farming or hunting. To test the authenticity of the commercial labels of meat products in the local market, we obtained DNA sequence information from 146 samples (14 beef and 132 game labels) for barcoding cytochrome c oxidase subunit I and partial cytochrome b and mitochondrial fragments. The reliability of species assignments were evaluated using BLAST searches in GenBank, maximum likelihood phylogenetic analysis and the character-based method implemented in BLOG. The Kimura-2-parameter intra- and interspecific variation was evaluated for all matched species. The combined application of similarity, phylogenetic and character-based methods proved successful in species identification. Game meat samples showed 76.5% substitution, no beef samples were substituted. The substitutions showed a variety of domestic species (cattle, horse, pig, lamb), common game species in the market (kudu, gemsbok, ostrich, impala, springbok), uncommon species in the market (giraffe, waterbuck, bushbuck, duiker, mountain zebra) and extra-continental species (kangaroo). The mountain zebra Equus zebra is an International Union for Conservation of Nature (IUCN) red listed species. We also detected Damaliscus pygargus, which is composed of two subspecies with one listed by IUCN as ‘near threatened’; however, these mitochondrial fragments were insufficient to distinguish between the subspecies. The genetic distance between African ungulate species often overlaps with within-species distance in cases of recent speciation events, and strong phylogeographic structure determines within-species distances that are similar to the commonly accepted distances between species. The reliability of commercial labeling of game meat in South Africa is very poor. The extensive substitution of wild game has important implications for conservation and commerce, and for the consumers making decisions on the basis of health, religious beliefs or personal choices. Distance would be a poor indicator for identification of African ungulates species. The efficiency of the character-based method is reliant upon availability of large reference data. The current higher availability of cytochrome b data would make this the marker of choice for African ungulates. The encountered problems of incomplete or erroneous information in databases are discussed.

Long-term RNA persistence in postmortem contexts.

Abstract: Ribonucleic acids (RNA) are generally considered fragile molecules that are readily degraded. However, there is growing documentation of long-term (from days to centuries) RNA persistence in a variety of contexts and tissue types, and as such a number of academic disciplines are beginning to exploit degraded RNA. While the reasons for its survival are not fully understood, there are several plausible mechanisms that would safeguard this molecule against degradation. However, after examining the literature available on the postmortem instability and decay mechanisms of RNA, it has become clear that limited experimental studies and no reviews offer an overview of these mechanisms. Hence in this review we outline molecular reasons for RNA surviving long-term postmortem, and provide specific examples of RNA survival in forensic, archival and archaeological contexts. A better understanding of the mechanisms of RNA decay will be crucial for developing expectations on its long-term survival.

Targeted sampling of cementum for recovery of nuclear DNA from human teeth and the impact of common decontamination measures.

Abstract: Background Teeth are a valuable source of DNA for identification of fragmented and degraded human remains. While the value of dental pulp as a source of DNA is well established, the quantity and presentation of DNA in the hard dental tissues has not been extensively studied. Without this knowledge common decontamination, sampling and DNA extraction techniques may be suboptimal. Targeted sampling of specific dental tissues could maximise DNA profiling success, while minimising the need for laborious sampling protocols and DNA extraction techniques, thus improving workflows and efficiencies. We aimed to determine the location of cellular DNA in non-degraded human teeth to quantify the yield of nuclear DNA from cementum, the most accessible and easily sampled dental tissue, and to investigate the effect of a common decontamination method, treatment with sodium hypochlorite (bleach). We examined teeth histologically and subsequently quantified the yield of nuclear DNA from the cementum of 66 human third molar teeth. We also explored the effects of bleach (at varying concentrations and exposure times) on nuclear DNA within teeth, using histological and quantitative PCR methods.

The truth is out there

Abstract: Here’s a mysterious menagerie for you: the Omani owl, the hero shrew, Mondolfi’s four-eyed opossum, the Annamite striped rabbit, and the Khasian leaf-nosed bat. You’re unlikely to encounter even one of these curious critters, as they’re all rare and highly localised, and you’ll certainly never see them together in one zoological collection. What unites them is that they are all ‘new species’ , discovered and named in the last few years. None have made headline news, it’s true, but certainly more of a splash than the enormous supporting cast of novel beetles, arriving in an unending procession, but sadly ignored by all but the most dedicated entomologist. Despite the thoroughness with which our own species already seems to have scoured the planet, new kinds of non-human inhabitants turn up all the time, thanks to intrepid zoological explorers and taxonomists, and aided by DNA-based analysis to demonstrate their positions in the tree of life. Such work is laborious, but luckily there’s another simpler way to find a new species – split an existing one. For example, twenty years ago there was only one species of orangutan, with Sumatran and Bornean populations designated subspecies; now these have official species status. Such decisions can have important and beneficial impacts on conservation efforts, particularly when, as with the unfortunate orangutans, or other newly discovered primates such as the Arunachal macaque, Caqueta titi monkey and Highland mangabey, they are highly endangered by habitat destruction and hunting. As if all this wonderful real life weren’t enough, some people have a need to believe in some less tangible things. Setting aside the clearly supernatural, from angels to zombies via fairies and trolls, there is a host of hypothetical animals that have been claimed to lurk out there – the creatures of cryptozoology. Encyclopaedist George Eberhart sets down some criteria for what constitutes a ‘cryptid’ [1], and classifies them into groups. First, some threshold of significance needs to be met – they must be “big, weird, dangerous, or significant to humans in some way”, and this therefore excludes the humble beetle. Also, some controversy needs to attach to their status, such as vociferous claims for their existence, combined with equally vociferous claims that they are imaginary. Among Eberhart’s cryptid categories are distribution anomalies, in which a known species is reported well outside its normal range; these include British sightings of panthers [2]. More interesting are the alleged survivals of extinct species, including contemporary reports of the proverbially dead dodo, and the famous Loch Ness Monster, claimed by some to be a plesiosaur species that miraculously survived the Cretaceous–Tertiary extinction event, 66 million years ago. Loch Ness watchers have produced a series of blurred black and white photographs that are claimed either to be the monster, or less exciting things, including the wake of a boat, a dog swimming towards the camera carrying a stick, and a model dinosaur head attached to a toy submarine. This raises the difficult issue of hoaxes – the North American legend of the jackalope, a jack-rabbit with antlers, led many creative taxidermists to get to work. Among cryptids that have no parallel in the fossil record or in living species are a number of humanoid creatures that perhaps lurk in the woods and mountains; these include the American bigfoot (also known as sasquatch), the Himalayan yeti, and the Australian yowie. Stories abound, and in the case of bigfoot are bolstered by photos of suitably large footprints, and grainy images of a creature that some infidels insist is a man in a gorilla suit. A video (available on YouTube) shows either a dozing bigfoot, or a person asleep under a rug, depending who you ask. Late last year a scientific paper appeared [3] claiming DNA-based analysis of bigfoot specimens, including whole-genome sequences. Don’t bother searching for it in PubMed, though, because it was published as the first and only paper in an online journal called De Novo[4]. Initially, the paper was sent to Nature, and also the Journal of Advanced Zoological Exploration in Zoology, which appears to be another pop-up journal [5]. If you are interested in the details, you can read the paper itself, plus reviewers’ comments and the authors’ responses [6], and make up your own mind about the robustness or otherwise of their claims. The methods used sound good, including sequencing of the entirety of the maternally-inherited mitochondrial DNA (mtDNA), and even the use of next-generation sequencing to analyse whole genomes in three cases. The DNA sources are 113 assorted samples of hair, blood, mucus, toenail, bark scrapings, saliva and skin with hair and subcutaneous tissue attached, contributed by a large number of bigfoot enthusiasts. And what of the data? Mitochondrial analysis showed only human sequences, belonging to a number of types (haplogroups) typical of the modern US population. Other sequencing indicated that “the species possesses a novel mosaic pattern of nuclear DNA comprising novel sequences that are related to primates interspersed with sequences that are closely homologous to humans.” However, there’s no attempt to focus on these ‘novel sequences’ and figure out what they might be. On the basis of a confusing mass of data and a certain amount of evolutionary naivety, the authors conclude that “the data conclusively proves that the sasquatch exist as an extant hominin and are a direct maternal descendent (sic) of modern humans.” It would be exciting if this were true, but unfortunately, it seems more likely that the samples are a mix of modern human DNA and some animal DNA or other. The United States is a populous and technologically advanced place, home to over 327 million mobile phones, so it’s puzzling that the bigfoot photos continue to be of such poor quality. And where do these creatures go when they die? A properly examined corpse would go a long way to silencing the sceptics. The Himalayas, on the other hand, are remote and relatively unpopulated, so it seems possible that yetis may actually live (and die) there undetected. Enter Bryan Sykes, himself something of a semi-mythical hominin in the scientific community since his retirement from the hurly-burly of standard research endeavour almost a decade ago. He has been in the media over the last few weeks describing his yeti project – like the bigfoot folks, he apparently sourced alleged yeti samples from anyone who was interested in contributing them. This is a worry, particularly given the possibility of mischievous hoaxers. Mitochondrial sequences apparently resemble those found in 120,000-year-old polar bear samples from Svalbard [7], leading to the suggestion that yetis are some kind of long-surviving bear species. This seems plausible, but it’s not yet published – Sykes says it will be, but hopefully not in De Novo.

Purification of crime scene DNA extracts using centrifugal filter devices

Abstract: Background: The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods: Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results: Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. Conclusions: Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used.

Metabarcoding avian diets at airports: implications for birdstrike hazard management planning

Abstract: Wildlife collisions with aircraft cost the airline industry billions of dollars per annum and represent a public safety risk. Clearly, adapting aerodrome habitats to become less attractive to hazardous wildlife will reduce the incidence of collisions. Formulating effective habitat management strategies relies on accurate species identification of high-risk species. This can be successfully achieved for all strikes either through morphology and/or DNA-based identifications. Beyond species identification, dietary analysis of birdstrike gut contents can provide valuable intelligence for airport hazard management practices in regards to what food is attracting which species to aerodromes. Here, we present birdstrike identification and dietary data from Perth Airport, Western Australia, an aerodrome that saw approximately 140,000 aircraft movements in 2012. Next-generation high throughput DNA sequencing was employed to investigate 77 carcasses from 16 bird species collected over a 12-month period. Five DNA markers, which broadly characterize vertebrates, invertebrates and plants, were used to target three animal mitochondrial genes (12S rRNA, 16S rRNA, and COI) and a plastid gene (trnL) from DNA extracted from birdstrike carcass gastrointestinal tracts. Over 151,000 DNA sequences were generated, filtered and analyzed by a fusion-tag amplicon sequencing approach. Across the 77 carcasses, the most commonly identified vertebrate was Mus musculus (house mouse). Acrididae (grasshoppers) was the most common invertebrate family identified, and Poaceae (grasses) the most commonly identified plant family. The DNA-based dietary data has the potential to provide some key insights into feeding ecologies within and around the aerodrome. The data generated here, together with the methodological approach, will greatly assist in the development of hazard management plans and, in combination with existing observational studies, provide an improved way to monitor the effectiveness of mitigation strategies (for example, netting of water, grass type, insecticides and so on) at aerodromes. It is hoped that with the insights provided by dietary data, airports will be able to allocate financial resources to the areas that will achieve the best outcomes for birdstrike reduction.

Clinal distribution of human genomic diversity across the Netherlands despite archaeological evidence for genetic discontinuities in Dutch population history

Abstract: Background The presence of a southeast to northwest gradient across Europe in human genetic diversity is a well-established observation and has recently been confirmed by genome-wide single nucleotide polymorphism (SNP) data. This pattern is traditionally explained by major prehistoric human migration events in Palaeolithic and Neolithic times. Here, we investigate whether (similar) spatial patterns in human genomic diversity also occur on a micro-geographic scale within Europe, such as in the Netherlands, and if so, whether these patterns could also be explained by more recent demographic events, such as those that occurred in Dutch population history.

Modeling the contrasting Neolithic male lineage expansions in Europe and Africa

Abstract: Background Patterns of genetic variation in a population carry information about the prehistory of the population, and for the human Y chromosome an especially informative phylogenetic tree has previously been constructed from fully-sequenced chromosomes. This revealed contrasting bifurcating and starlike phylogenies for the major lineages associated with the Neolithic expansions in sub-Saharan Africa and Western Europe, respectively.

DNA fingerprinting in anthropological genetics: past, present, future.

Abstract: In 1985, Sir Alec Jeffreys developed the variable-number tandem repeat method used to identify individuals and giving researchers the first DNA fingerprints. These initial methods were used in anthropological genetics, a field that uses a comparative approach to answer questions about human history, including the discernment of the origin of Native American populations and the discrimination of clan affiliation from individuals in Siberia. The technological and methodological advances since this time have led to the use of many more markers, including restriction fragment length polymorphisms, Y chromosomal and autosomal short tandem repeats, single nucleotide polymorphisms, and direct sequencing not only to identify individuals, but to examine frequencies and distributions of markers (or “prints”) of entire populations. In the field of anthropological genetics these markers have been used to reconstruct evolutionary history and answer questions concerning human origins and diaspora, migration, and the effects of admixture and adaptation to different environments, as well as susceptibility and resistance to disease. This review discusses the evolution of DNA markers since their application by Sir Alec Jeffreys and their applications in anthropological genetics.

The man behind the DNA fingerprints: an interview with Professor Sir Alec Jeffreys

Abstract: In this interview we talk with Professor Sir Alec Jeffreys about DNA fingerprinting, his wider scientific career, and the past, present and future of forensic DNA applications. The podcast with excerpts from this interview is available at: http://www.biomedcentral.com/biome/alec-jeffreys .

Major historical dietary changes are reflected in the dental microbiome of ancient skeletons

Abstract: The post-industrial lifestyle has many disadvantageous effects on our health. One of the factors is modern nutrition, which has been associated with epidemic burdens, such as obesity and cardiovascular diseases. At least two major shifts have occurred in the nutritional history of humans: the use of carbohydrate-rich diets which were adopted around 10,000 years BP due to Neolithic farming, and later the influence of industrially processed flour and white sugar after the industrial revolution in the 1850s. In a recent paper in Nature Genetics Adler et al. used a novel approach to see how these dietary changes affected the oral microbiome by analyzing the ancient microbial DNA in the calcified dental plaque from 34 early European skeletons.

Flogging a dead horse

Abstract: We may live in a global village, and do our research in multinational departments, but local cultural traditions still count. A chilly October morning in the bleak English Midlands, and a PhD student newly arrived from Italy (let’s call her Giovanna) takes a cigarette break outside our building. In the camaraderie of smokers, she strikes up a conversation with Keith, who is taking a rest from the autoclaving. “Where can I find horse?”, she asks. Processing this odd question, via her foreign-ness, he pictures Leicester race-course, and wonders about country riding clubs. But he is wide of the mark. Giovanna’s thoughts are in the kitchen, and when Keith realizes this, the conversation stops short. For the British (and for most of the English-speaking world), eating horsemeat (hippophagy) is just not an option, something they would never, ever do. At least, that’s what we all thought until recently, when it turned out that many of us had been unwittingly doing exactly that for some time. The ‘horsemeat crisis’ has moved from the detection of a few percent contamination in burgers to the finding of lasagne purportedly made with beef, but actually being pure, unadulterated horse. The health risks are probably minimal – there is some concern about a drug, phenylbutazone, allowed in horses but prohibited in humans – but nonetheless many tonnes of processed foods have been destroyed. According to Alan Davidson’s compendious and wonderful Oxford Companion to Food[1], the most avid consumers of horsemeat are the Italians, particularly those from the region around Venice, but there is also considerable enthusiasm among the peoples of France, Belgium, the Netherlands, Germany, Sweden and Iceland. A dislike of horsemeat is one of those things (along with a similar feeling about frogs and snails) that distinguishes a true Brit from a Frenchman – what some have called ‘gastro-nationalism’. To the novice British tourist in France a boucherie chevaline, the specialist horse-butcher marked by a gilded horse-head above the door, is a surprising sight. Interestingly, given the current crisis, the reason that these were kept distinct from other butchers (bouchers classiques) was to guard against the threat of fraudulent sale of horse as beef. Revulsion at eating horses is partly connected to the idea of the horse as a noble animal, but also to the more pragmatic point that, as a means of transport and traction, it was traditionally worth more alive than dead. In medieval times eating horses was a desperate act associated with famine, so a link was formed between hippophagy and poverty. Nineteenth-century attempts to turn British attitudes around [2] included a proposal to rename horsemeat cheval, in the same euphemistic way that we use words derived from French terms for the meat of the pig (porc) and cow (boeuf). In 1868 a horse banquet was held for 150 guests by London’s Society for the Propagation of Horse Flesh as an Article of Food. Despite all this, it didn’t catch on. Apart from the general dislike, a particular problem may have been that British horsemeat came from tough old animals at the end of their working lives. Finding the horse in your burger requires DNA analysis, and exploits the marked interspecies differences between mitochondrial DNAs (mtDNAs) that result from their relatively high mutation rates. The properties that have made this molecule a popular tool in animal species ‘barcoding’ projects [3] also allow the design of real-time polymerase chain reaction (PCR) assays that specifically identify horse DNA in a mixture of meats from other species. These assays are remarkably sensitive, allowing the detection of levels as low as 0.0001% [4]. The journal Meat Science has plenty of articles on this topic, including a nice sausage-specific example [5]. Such studies also describe the identification of a wide range of species mixtures other than horse and cow, including chicken, turkey, sheep, pig, and donkey. Distinguishing horse from donkey can be tricky, as the species are closely related, but apparently important, because some cultures eat one, but not the other. The Italians are fond of both. However, as well as the sausage manufacturers, horses (Equus caballus) and donkeys (Equus asinus) can do the mixing themselves, to produce interspecific hybrids - mules and hinnies. The nature of the offspring depends on the sex of the parents, and a mnemonic helps here: for a mule, the mare is the mother; for a hinny, the he is a horse. Producing mules (the more common and useful hybrid) is not an easy business, because, ‘in equine courtship it is the stallion who takes the initiative, and… the donkey stallion is a fastidious fellow, and he does not willingly engage in anything which might be regarded as slightly improper’ [6]. The phenotypes are different (probably as a result of parentally imprinted genes), with a mule appearing to have a donkey’s ears and a horse’s tail, and a hinny the other way round. People eat mules, as well as donkeys and horses, and in meat contamination testing, mule meat would appear to be horsemeat, because of the maternal inheritance of mtDNA. Horses have 64 chromosomes, and donkeys 62, so mules and hinnies carry the intermediate odd number 63, which leads to infertility in the hybrids because oocytes fail during meiosis. As Thomas Bewick writes in his A General History of Quadrupeds[7], ‘Nature has providently stopped the further propagation of these heterogeneous productions, to preserve, uncontaminated, the form of each animal; without which, …every creature, losing its original perfection, would rapidly degenerate’. Occasionally, though, a female mule produces a foal after mating with a male donkey; so rare is this event that the Romans used the phrase Cum mula peperit (when a mule gives birth), equivalent to the English ‘once in a blue moon’. One such product, a healthy female in China who could plough a field by herself at 4 years of age, had 62 chromosomes, a mix of horse and donkey with a bias towards the latter [8]. In the global market-place of the internet, obtaining novel and exotic animal food products is becoming ever easier, and if you have a strong stomach and wonder how broad the scope is, just visit http://www.exoticmeatmarkets.com, where you’ll find pretty much everything (except horse…). But are these things really what they say? Contributors to Meat Science, take note – there is work for you to do! Back in our multinational department, too, the gastro-exotica goes on, as my Estonian post-doc kindly brings me a present: a tin of karuliha – bear-meat. We have yet to eat it.

Comment on Kokshoorn, B, and Blankers, BJ ‘Response to Grisedale, KS and van Daal, A: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method’

Abstract: Kokshoorn and Blankers responded to our recent article by saying that replicate analysis and consensus profiling of low template samples was best in terms of reliability and objectivity. We agree that the consensus approach has benefits, particularly in eliminating non-repeating spurious alleles from the final profile. However, with the development of statistical models that can accommodate stochastic effects and allele drop in, it may be beneficial to perform a single amplification with three times the amount of template, since much information is lost from the profile using the consensus approach.

Curiosity in the genes: the DNA fingerprinting story

Abstract: It is unusual for a scientific field to be associated with a single individual, but in the case of the subject of the thematic series now being launched in Investigative Genetics, this is surely so; Alec Jeffreys (Figure 1) is DNA fingerprinting. Having invented the method, he coined the perfect name for it - how different things might have been if it had been called the tandem-repeat-based identification technique (or something similarly dull). He realized its potential and immediately applied, developed and refined it. He then followed his nose to unravel the mystery of the madly mutable minisatellites that make up DNA fingerprints, and eventually to understand the engines of genome variability that reside in recombination hotspots. 'I think I was born a scientist’, he has said [1]. He certainly seems to have been born with unquenchable enthusiasm and curiosity, and it is this quality that has led him on his extraordinary scientific journey. Figure 1 Professor Sir Alec Jeffreys. (Picture taken by Colin Brooks, courtesy University of Leicester) Prof Sir Alec Jeffreys has provided informed consent for the publication of his photograph. The story of DNA fingerprinting has been told more than once, but that is because it is such a good tale (its inventor tells it very well himself [2]), and it deserves a brief retelling here. Having noticed the sequence similarity between core elements of tandem repeats in the myoglobin gene and a few other known minisatellites, Alec made a pure repeat probe, and radiolabelled and hybridized it to Southern blots of restriction-digested DNA. The probe cross-reacted with a set of hypervariable minisatellites, and on the morning of Monday 11 September 1984 the first fuzzy DNA fingerprint emerged from the developing tank. In this 'eureka moment’ , Alec could immediately see the diversity, and the pattern of inheritance in DNA from a human pedigree. In his seminal paper [3] he foresaw roles for the method in linkage analysis, in testing tumour clonality, twin zygosity and paternity, in forensic typing, and in dissecting fundamental aspects of mutation and recombination processes. In one remarkably productive year these ideas were developed in four further papers [4-7], three of them in Nature. The first DNA fingerprinting application was in parentage testing [6]; normally it is the father who is in doubt, but this unusual and challenging case was a maternity test, with paternal DNA unavailable. British nurse Christiana Sarbah’s 13-year-old son Andrew was denied re-entry to the United Kingdom after a visit to Ghana, the immigration authorities suspecting that he was not her child. Given three undisputed children for comparison, it was possible to reconstruct the absent father’s DNA fingerprint, and to strongly support the claimed maternity over alternative relationships such as aunt-nephew - something that was not achieved with traditional protein polymorphisms such as blood groups. In an immigration tribunal, the UK Home Office accepted the DNA evidence, and allowed Andrew to stay with his mother and siblings. It also stated that it would not contest future immigration disputes where similar evidence was available, which effectively broke a log-jam of such cases, but created an avalanche of casework for the Jeffreys lab before the methods were commercialized [8]. Hot on the heels of this came the first application of DNA fingerprinting in forensic identification, in a case that beautifully exemplifies the power of DNA evidence to link crime-scenes, to exclude suspects, and to support convictions. Work with Peter Gill and Dave Werrett had shown that DNA fingerprints could be obtained from old samples, and importantly that the method of differential lysis could yield male-specific information from mixed rape-case samples [4]. When Leicestershire Police suggested DNA fingerprinting be applied in a local murder investigation it seemed straightforward - two 15-year-old girls had been raped and strangled about 3 years apart with the same modus operandi, and a suspect was in custody who had confessed to the second killing. As expected, DNA profiles (now based on specific cloned minisatellites, known as single-locus probes) from semen samples at both crime-scenes showed that the same man was responsible in each case. The surprise, though, was that the suspect matched neither scene - the first DNA-based exoneration. A bold police decision then triggered the first DNA-based mass screen to find the true culprit. Blood samples were taken from 5,000 local men, and following initial exclusion using protein polymorphisms, the remaining 500 were tested using the new DNA technology. None matched, but the stalemate was broken when Ian Kelly, a colleague of the perpetrator Colin Pitchfork, told friends that he had been persuaded to provide a blood sample on his workmate’s behalf. The eventual DNA profile from Pitchfork himself matched the crime-scenes, he was convicted, and he remains incarcerated today. As well as human DNA, that first autoradiograph had included samples from various other species, and showed that the core minisatellite probes also detected hypervariable loci in non-human genomes [1]. Applications promptly followed in mice [9], cats and dogs [10], and birds [11,12]. Perhaps the last hurrah of true DNA fingerprinting came with the autoradiograph published to confirm that Dolly the sheep was indeed a clone [13]. I first encountered Alec when he gave a barn-storming talk to the 1991 International Congress of Human Genetics, in Washington DC. His subject there, described with his trademark enthusiasm, was a near-magical trick that detected and mapped the sequence variation between individual repeat units within a minisatellite, revealing a mind-boggling degree of diversity. He had embraced PCR early, showing that profiling could be done from trace amounts of DNA [14], then developing his improbable method - minisatellite-variant-repeat (MVR) PCR [15]. With a Y-chromosomal minisatellite in hand [16], I wanted to try it too, and came to Leicester in 1992, finding the same hospitable environment that Alec discovered fifteen years earlier, and, in Alec himself, a generous sponsor. MVR-PCR was perhaps the ultimate DNA fingerprint, but instead of impacting on forensic analysis, it turned out to be a key tool in understanding the complex recombination processes that drive minisatellite diversity [17]. Forensic DNA testing was moving towards short tandem repeats (STRs), and after using these markers in a collaboration with Erika Hagelberg to identify the skeletal remains of a murder victim [18], and of Josef Mengele [19], Alec’s research interests shifted away from forensics. He has, however, maintained his willingness to engage in public debate about forensic genetics and DNA databases. Alec himself deplores the bean-counting that goes on in judging science these days, but nonetheless it is worth noting that he has well over 200 publications, an h-index of 67, and over 21,000 citations of his work, a figure that continues to grow. Since joining the University of Leicester in 1977, he has enhanced the reputation of the place enormously, as can be confirmed by a casual glance at almost any piece of University publicity. It was something of a surprise when he retired in September 2012 since his enthusiasm for experimental science and his continuing technical elan seemed likely to keep him at the bench, pipette in hand, forever. Our Vice-Chancellor, during the valedictory address, noted the irreplaceability of Alec, but said that he took some comfort from the fact that 'at least we now have Richard III’ [20]. The amusement expressed by Alec at being supplanted by the skeletal and wormy [21] remains of a long-dead king can be imagined. DNA fingerprinting has also, of course, had a massive impact on society. Indeed, it is hard to think of another modern scientist whose work has had the societal reach of Alec’s. His list of prizes and honorary degrees is almost absurdly long (particularly when set beside those of his departmental colleagues), but it is the public recognition that is particularly telling - Midlander of the Year (1989), Honorary Freeman of the City of Leicester (1993), and Morgan Stanley Greatest Briton (2007). Another honour that many UK celebrities quietly crave is an appearance on BBC Radio 4′s long-running programme Desert Island Discs. Participants must choose eight records they would take to a desert island and use them to punctuate an interview about their lives; it is said that many a deceased public figure is found to have their disc list stored safely away in preparation for the call. Alec was called [22], and his appearance shook middle England out of its slumber by including Feel the Beat, by Trance DJ 'Darude’ , among his chosen records. As well as a spectacular researcher, Alec has been a highly valued colleague in Leicester - a modest and truly collegiate person who has been a popular teacher of undergraduates, who has shown an almost superhuman ability to unfailingly ask a pertinent question at the end of a seminar (no matter how boring or impenetrable it was), and who has been generous with his advice and support. These days our universities, institutes and funders spend a lot of time beating us over the head with demands for our research strategies, translational research plans, and pathways to impact. As Alec has said [23], 'If someone had told me in 1980, 'Go away and figure out a way of identifying people with DNA’ , I would have sat there looking very stupid and got nowhere at all’. The story of DNA fingerprinting is a reminder that following your nose, and your scientific enthusiasm, can get you a long way.

Editors' pick: transcriptomes of 1000 genomes.

Abstract: Next generation sequencing (NGS) technologies facilitate massive human DNA sequence variation data to be produced in a remarkable manner and speed. The functional effects of these variants can now also be analyzed by high-throughput RNA sequencing (RNA-seq) of the transcriptome. Prior to RNA-seq, analysis of human gene expression was performed by expression arrays with more limited coverage. In the recent issue of Nature, Lappalainen et al.[1] have, for the first time, used the full capacity of RNA-seq to create the largest existing catalog of potential causative functional variants in the genome and to characterize transcriptome variation in human populations in a subset of individuals from the 1000 Genomes Project. They reported a deep analysis of high-quality mRNA and miRNA sequences from lymphoblast cell lines from > 450 individuals belonging to five populations (CEPH-CEU Europeans, Finns, British, Toscani Italians, and Yoruba from Nigeria). The authors addressed several questions with their dataset. First, they investigated human transcriptome variation at the population level, which can manifest in overall expression levels or in splicing (in one gene). In a genome-wide perspective, Lappalainen et al. showed that population differences account for about 3% of the total variation, but they also identified 263–4.379 genes with differential expression and/or transcript ratios between population pairs. In the pairwise analysis, they observed something interesting: the African-European population pairs had a higher contribution of genes with differential splicing (75 to 85%) than the European populations had between each other (6 to 40%). Although this phenomenon has not been previously observed in humans, it is in agreement with phylogenetic differences between species being better captured by splicing rather than expression levels. Lappalainen et al. analyzed 644 autosomal miRNAs that could be quantified from > 50% of the individuals. Sixty of those miRNAs (9.3%) had significant cis-expression QTLs (eQTLs) for miRNA expression levels, indicating that genetic effects on miRNA expression are more widespread than had been known. The authors also provide evidence for the existence of feedback loops for mRNA and miRNA genes to have an effect on each other’s expression supporting the idea that miRNAs offer robustness in the expression programs. Altogether, Lappalainen et al. have produced the largest and most diverse catalogue of cis-regulatory variants in a single tissue to date. The authors used their data to analyze the functional properties of the newly found set of regulatory variants and transcriptome effects of protein-truncating loss-of-function variants. This collaborative effort further used the potential of RNA-seq to discover regulatory variants that affect not only expression levels but also splicing. Of the 7.825 genes with expression QTLs (eQTLs), 34% have a second, independent eQTL for any of their exons. The authors concluded that in the transcriptome there exists allelic heterogeneity for regulatory effects on a single gene and independence of exons within the same gene. As an ancillary benefit, the authors demonstrated that RNA-seq data is consistent even when produced in different laboratories, with more detailed analysis in a companion paper in Nature Biotechnology[2]. Namely, in the replicate analyses of RNA-seq data from seven laboratories, there was a smaller amount of variation among the laboratories than seen among the individuals. The study is a great example of integration of genomic sequencing and cellular phenotype data. Obviously, the amount of data produced in this study is enormous, and it can easily be expected that the data will also be used to support other pivotal work. Meanwhile, it will take some time to completely digest the magnitude of data and appreciate the impact it will have on functional genomics.

Editors' pick: re-'colon'-ization of healthy microbiota after recurrent C. difficile infection.

Abstract: The story of Kaitlin Hunter [1] brings to light the potential triumphs of the age of biotechnology and how an understanding of the interplay of genomics and the human microbiome can provide a cure to a life-threatening disease. After a serious car accident and under standard healthcare, Kaitlin was given antibiotics to prevent infection. Likely, the antibiotic treatment compromised her normal gastrointestinal flora and permitted Clostridium difficile, a toxin-producing, Gram-positive, anaerobic, spore-forming bacillus, to become established. C. difficile causes diarrhea and is linked to approximately 14,000 deaths each year in the United States alone [2]. However, a novel solution was undertaken to treat Kaitlin - her mother, a healthy donor, provided a fecal sample which was transplanted into Kaitlin’s colon. Her mother’s bacteria re-colonized the colon and Kaitlin was cured. This life-saving treatment may make one reconsider the phrase of ‘taking no “_ _ _ _” from anyone’. While a fecal transplant makes logical sense for staving off a C. difficile infection, there are patients (and physicians) who may not opt for such a treatment. First, patients may not relish the thought of receiving fecal matter that would be infused directly into their colons. The thought of it is unpleasant at best. Second, a fecal sample even from a healthy donor would be replete with bacteria, some of which may be pathogenic or at least opportunistic pathogens. However, Petrof et al. [3] have devised a solution to ‘RePOOPulate’ the intestines of patients with C. difficile infections. They used investigative genetics technology to develop a stool substitute, which poses less risk to the patient, and probably for the first time employed massively parallel sequencing (MPS) to track a transplant and monitor a patient’s recovery. Thereby, they demonstrated that the disruptive MPS technology has immediate diagnostic applications. Petrof et al. obtained a stool sample from a healthy, 41-year-old woman and isolated 62 different bacteria. After rejecting antibiotic-resistant microbes - those that may cause disease, 33 intestinal bacteria isolates remained. These isolates were reconstituted in proportions to approximate those from the original stool sample. Thus Petrof at el. created a stool substitute mixture comprised of a multispecies community no longer in the presence of fecal material. Two patients were selected for ‘repoopulation’. They were a 74-year-old Caucasian woman with six episodes of recurrent C. difficile infection over an 18-month period and a 70-year-old Caucasian woman with a history of peripheral neuropathy, which predisposed her to recurrent skin and soft tissue infections and under cefazolin treatment developed a C. difficile infection. Both were infected with a particularly hypervirulent strain of C. difficile, ribotype 078. These patients posed a challenge because of age and recurrent infection. The status of pretreatment and post-treatment microbiota was monitored by sequencing PCR products of the bacterial V6 rRNA region on the Ion Torrent platform with low to medium throughput chips for the instrument. The Ion Torrent is one of the commercially available massively parallel sequencers that enables genomic analyses on the bench top of most laboratories which was only a short time ago the domain of large genomic centers. Throughput of the sequencing chips is upwards of 100 megabases and for single patient monitoring was deemed sufficient to obtain a depth of coverage to assess diversity of the patients’ intestinal microbial flora. Up to 12 samples were multiplexed on each chip through use of indexing. Therefore community microbial composition analysis could be assessed in virtually real-time (that is, 24 to 48 h) to track the success or failure of the stool substitute transplant to re-establish the microbiota of the patients. Sufficient V6 rRNA reads were obtained (between 3,758 and 76,752 V6 rRNA reads per sample for Patient 1 and between 19,751 and 64,200 reads per sample for Patient 2) to estimate general and relative diversity of the microflora of the patients. Within 2 to 3 days of stool substitute treatment, each patient recovered a normal bowel pattern and remained symptom-free for up to 6 months when monitoring was stopped. The first patient displayed a highly diverse microbiota that after treatment became less diverse and after 6 months regained similar diversity as that of pretreatment. The second patient’s microbiota had relatively low diversity and became more diverse following treatment which stabilized with a higher diversity than that of pretreatment. Genomic analyses demonstrated that the rRNA sequences representative of the stool substitute were infrequent in the pretreatment stool samples of the patients. Once stabilized, the same sequences comprised >25% of the patients’ samples. Taxonomic assignment with rRNA V6 region could only be carried out at the family level as greater phylogenetic resolution cannot be attained using a single house-keeping gene. This lack of resolution did not compromise monitoring for stool substitute transplantation success. However, given the throughput of MPS, adding a few more housekeeping genes could easily provide species/strain level resolution and even monitor for mutation that may arise. Although successful, the RePOOPulation study was limited to two patients and more studies are needed. However, Petrof et al. have demonstrated the proof of concept that this stool substitute, synthetically reconstituted from fecal material, can be an effective treatment for C. difficile infection, particularly for recurrent infections with hypervirulent strains. The authors point out that there are several advantages to using a substitute over actual fecal material, besides the obvious distaste of having such material injected into one’s body. These include ‘the exact composition of bacteria administered is known and can be controlled; the bacterial species composition can be reproduced, should a future treatment be necessary; preparations of pure culture are more stable than stool, which some groups recommend should be collected fresh and instilled into the recipient within 6 h of collection; an absence of viruses and other pathogens in the administered mixture can be ensured, thereby improving patient safety; and the administered organisms can be selected based on their sensitivity to antimicrobials, allowing an enhanced safety profile’. It is hard to believe that only a few years ago the human microbiome was defined solely by E. coli and bacteria that caused bad breath and tooth decay. The diversity of the human microbiome is substantial and its secrets, as they unravel, promise to bring additional insights to human health and potential treatments to improve well-being. Investigative genetics tools, such as high throughput sequencing and targeted gene diagnostics, will figure prominently into our personalized medical treatment and fecal, or simulate, transplantation is just the tip of the iceberg. Who knows… the next time one of us travels to an area of the world where the microflora of the water can cause intestinal discomfort, a possible pretreatment for better well-being during travel may be a stool simulant enema.

Editors’ Pick: mad and genius in the same gene?

Abstract: In the current world of genetic publishing and multi-author papers, where sometimes it is possible that several hundreds of co-authors are listed on a single article (and yes, I am thinking of genome-wide association studies here), I am always intrigued by single author papers. Hence, a not too recent paper by Szabolcs Keri from Semmelweis University in Budapest drew my attention, also because of the interesting title ‘Genes for psychosis and creativity’ [1]. This reminded me of the German saying, ‘Genie und Wahnsinn liegen oft dicht beieinander,’ which translates as: genius and madness are often at close quarters, and is similar, but not quite the same as the English phrase, ‘There is a fine line between genius and madness.’ Does this study provide one of the not so frequent cases where a layman observation that led to commonly used phrases, even in different languages, eventually receives scientific proof? Previous studies had suggested that the neuregulin I gene, which is involved in neuronal development, synaptic plasticity, glutamateric neurotransmission and glial function, serves as a candidate gene for psychosis [2]. In particular, the T/T genotype of the functional promoter polymorphism SNP8NRG243177 or rs6994992, which leads to increased neuregulin I gene expression, is associated with an increased risk of psychosis and other psychological and neurological phenotypes [3,4]. Keri investigated whether this particular DNA variant also has an advantageous effect. He studied 200 healthy volunteers with high intellectual performance as measured via a battery of tests and questionnaires, who were genotyped at the neuregulin 1 promoter DNA variant rs6994992. Grouping these 200 high intellectual achievers according to rs6994992 genotypes revealed that the T/T carriers had statistically significantly higher creativity measures compared with the C/C and the C/T carriers, with the C/T heterozygotes usually performing at an intermediate level relative to the T/T and the C/C homozygotes. No significant differences between individuals belonging to these three genotype groups were observed for other parameters tested, such as age, gender, education, socioeconomic status, intelligence quotient (IQ), employment, marriage, and so on. Two additional DNA variants from the neuregulin I gene that are not implicated in psychosis and do not affect gene expression were also tested and did not indicate similar associations. Based on these data, Keri argues that the neuregulin I gene, previously implicated in psychosis and altered brain structure and function, has a significant impact on human creativity. Is this evidence enough to support the conclusion that genius and madness are indeed related to each other because they are determined by the same gene? The author himself already admits some caveats in his study, such as that the findings were obtained from a selected group of people with high intellectual performance. It may be (as was not tested) that the observed effect is not found in an intellectually less-prominent sample. He also points out that in the general population rs6994992 is not associated with schizotypical traits as reported previously [5]. Furthermore, genetic evidence on the involvement of neuregulin 1 in psychotic conditions appears not without doubt. For instance, while some candidate gene studies reported the association of neuregulin-1 DNA variants with schizophrenia (for example [6]) and a genome-wide linkage analysis highlighted a candidate region for schizophrenia that includes the neuregulin-1 gene [7], several genome-wide association studies highlighted genes other than neuregulin-1 to be involved in schizophrenia (for example [8,9]). If Keri’s findings indeed reflect a general trend and if, indeed, the neuregulin 1 gene determines psychotic conditions, the question on how the rs6994992T/T genotype leads to higher creativity will be of interest. The author suggests that reduced cognitive inhibition, known to be related to schizotypical features and associated with increased creativity in people with high intelligence, may be the key here, but data on the direct role of rs6994992 in reduced cognitive inhibition is missing thus far. Clearly, much further work needs to be done before scientific data allow us to conclude whether madness and genius indeed share biological determination and, thus, can be considered to be at close quarters, or not. Until this is achieved, we may keep using the saying ‘Genie und Wahnsinn liegen oft dicht beieinander,’ but need to be aware that scientific evidence clearly supporting it is still lacking.

ENCODE and its first impractical application

Abstract: The C value paradox, as initially coined, was encountered in early eukaryotic genomic studies with the oddity that genome size was not necessarily correlated with organism complexity [1]. With the discovery of non-coding DNA in the 1970s, it became apparent that the size of the eukaryotic genome was not related to the number of genes contained within it. Indeed, only a small portion (approximately 2%) of the human genome carries coding genes [2-4], the rest being the so-called “junk DNA” [5]. The human genome project further elucidated the number of genes in our genomes - counting a paltry 20,000 to 25,000 genes [2-4]. With so few genes one might ask “how could such a complex organism as Homo sapiens pass on the necessary genetic blueprint to the next generation?” An equally enticing question could be “how could nature be so wasteful and commit so much junk DNA to the human genome?” The Encyclopedia of DNA Elements (ENCODE) project has shed some light on these two questions. There is not one paper to cite but greater than 30 studies [6] that were coordinated and published in concert describing the results of a multi-year consortium effort to catalogue the functional elements of human DNA. Hundreds of authors reported on analyses of thousands of data sets. A good summary of the work is captured in the ENCODE Project Consortium’s September 2012 publication titled “An integrated encyclopedia of DNA elements in the human genome” [7]. The ENCODE project identified a large number of functional elements, defined as sites that encoded a product or exhibited a biochemical signature in the human genome. The power of current DNA sequencing technologies made the Consortium project possible. The depth of analysis is impressive. In this one paper more than 1,600 data sets were analyzed for a multitude of elements including human protein-coding and non-coding RNAs, pseudogenes, RNA from different cell lines, binding locations of a number of DNA-binding proteins and RNA polymerase components, DNase I hypersensitive sites, locations for histone modifications, and DNA methylation. The most exciting finding and one that may begin to address the two questions posed above was that 80.4% of the genome has a biochemical function, that is, it is covered by or near at least one ENCODE-identified element. More precisely, a large portion of the human genome contains a regulatory event. The authors state that “95% of the genome lies within 8 kilobases (kb) of a DNA–protein interaction…, and 99% is within 1.7 kb of at least one of the biochemical events measured by ENCODE.” The outcome is that the noncoding junk DNA is far from being useless genome filler. Instead, seemingly inert DNA can influence functional genes. The nature of genetic and epigenetic control is quite complex and exquisite and today all that more appreciated. ENCODE is a public resource that will contribute substantially to the understanding of gene expression and mechanisms of disease and, hopefully, cures. Surprisingly though, what might be the first application of ENCODE data is not directed toward improving human health through molecular biology. Bolstered by the newly found functional nature of a greater portion of the junk DNA, an appeal in a U.S. Court has been brought forward in part on a basis that information derived from typing the short tandem repeat (STR) markers used in forensic human identification worldwide violates an individual’s privacy [8]. The argument exploits ENCODE data to suggest that there is some noticeable predictive power hidden with the forensic STRs related to the health status of an individual. After all the Consortium publication suggests that “Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation.” Such ENCODE information should be understood and the limitations should be appreciated; we are far from extracting predictive power and unlikely to do so with the forensically-relevant STRs. Even without knowing any causal relationship, as now might be intimated by some with the ENCODE project data, association studies between the forensic STR loci and disease genes generally have come up empty, providing little if any predictive power. One could argue that one STR, the TH01 locus, has been shown to have some effect on expression at the gene Tyrosine Hydroxylase[9] well before the onset of the ENCODE project. Still armed with such information, there is little predictive power regarding an individual’s health status by knowing the allelic repeat state of the TH01 locus. Such limited power is not surprising; next generation sequencing and genome wide association studies overwhelmingly find that most diseases are genetically complex and one marker provides little value in determining risk of disease or outcome from therapeutics. It would be a shame that the phenomenal effort that brought forth ENCODE might be misused to attempt to breach the foundations of forensic DNA typing. ENCODE’s value is in laying a foundation of the intricate functionality of the human genome that someday may help improve the human condition. Certainly, claims of privacy violations via human identification by STR typing are unfounded and criticizing this powerful forensic tool, based on ENCODE data, does not improve the human condition.