Working with mitochondrial DNA from highly degraded samples is challenging. We present a whole mitogenome Illumina-based sequencing method suitable for highly degraded samples. The method makes use of double-stranded library preparation with hybridization-based target enrichment. The aim of the study was to implement a new user-friendly method for analysing many ancient DNA samples at low cost. The method combines the Swift 2S™ Turbo library preparation kit and xGen® panel for mitogenome enrichment. Swift allows to use low input of aDNA and own adapters and primers, handles inhibitors well, and has only two purification steps. xGen is straightforward to use and is able to leverage already pooled libraries. Given the ancient DNA is more challenging to work with, the protocol was developed with several improvements, especially multiplying DNA input in case of low concentration DNA extractions followed by AMPure® beads size selection and real-time pre-capture PCR monitoring in order to avoid cycle-optimization step. Nine out of eleven analysed samples successfully retrieved mitogenomes. Hence, our method provides an effective analysis of whole mtDNA, and has proven to be fast, cost-effective, straightforward, with utilisation in population-wide research of burial sites.

• MeSH
• analýza nákladů a výnosů MeSH
• genom mitochondriální * MeSH
• lidé MeSH
• mitochondriální DNA genetika   MeSH
• polymerázová řetězová reakce MeSH
• soudní genetika metody   MeSH
• starobylá DNA * MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Research on ancient and forensic DNA is related in many ways, and the two fields must deal with similar obstacles. Therefore, communication between these two communities has the potential to improve results in both research fields. Here, we present the insights gained in the ancient DNA community with regard to analyzing DNA from aged skeletal material and the potential use of the developed protocols in forensic work. We discuss the various steps, from choosing samples for DNA extraction to deciding between classical PCR amplification and massively parallel sequencing approaches. Based on the progress made in ancient DNA analyses combined with the requirements of forensic work, we suggest that there is substantial potential for incorporating ancient DNA approaches into forensic protocols, a process that has already begun to a considerable extent. However, taking full advantage of the experiences gained from ancient DNA work will require comparative studies by the forensic DNA community to tailor the methods developed for ancient samples to the specific needs of forensic studies and case work. If successful, in our view, the benefits for both communities would be considerable.

• MeSH
• DNA fingerprinting MeSH
• DNA * genetika   MeSH
• lidé MeSH
• nekrotická degradace DNA MeSH
• senioři MeSH
• soudní genetika MeSH
• starobylá DNA * MeSH
• Check Tag
• lidé MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

One of the main goals of the Spanish and Portuguese-Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. Due to this fact, GHEP-ISFG holds different working commissions that are set up to develop activities in scientific aspects of general interest. One of them, the Mixture Commission of GHEP-ISFG, has organized annually, since 2009, a collaborative exercise on analysis and interpretation of autosomal short tandem repeat (STR) mixture profiles. Until now, six exercises have been organized. At the present edition (GHEP-MIX06), with 25 participant laboratories, the exercise main aim was to assess mixture profiles results by issuing a report, from the proposal of a complex mock case. One of the conclusions obtained from this exercise is the increasing tendency of participating laboratories to validate DNA mixture profiles analysis following international recommendations. However, the results have shown some differences among them regarding the edition and also the interpretation of mixture profiles. Besides, although the last revision of ISO/IEC 17025:2017 gives indications of how results should be reported, not all laboratories strictly follow their recommendations. Regarding the statistical aspect, all those laboratories that have performed statistical evaluation of the data have employed the likelihood ratio (LR) as a parameter to evaluate the statistical compatibility. However, LR values obtained show a wide range of variation. This fact could not be attributed to the software employed, since the vast majority of laboratories that performed LR calculation employed the same software (LRmixStudio). Thus, the final allelic composition of the edited mixture profile and the parameters employed in the software could explain this data dispersion. This highlights the need, for each laboratory, to define through internal validations its criteria for editing and interpreting mixtures, and to continuous train in software handling.

• MeSH
• DNA fingerprinting normy   MeSH
• laboratoře statistika a číselné údaje   MeSH
• lidé MeSH
• mikrosatelitní repetice * MeSH
• pravděpodobnostní funkce MeSH
• software MeSH
• soudní genetika normy   MeSH
• společnosti vědecké * MeSH
• výzkumná zpráva normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.

• MeSH
• Diptera klasifikace   genetika   růst a vývoj   MeSH
• DNA izolace a purifikace   MeSH
• hmyzí proteiny genetika   MeSH
• reagenční diagnostické soupravy MeSH
• respirační komplex IV genetika   MeSH
• sekvenční analýza DNA MeSH
• soudní genetika metody   MeSH
• taxonomické DNA čárové kódování metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Geografické názvy
• Česká republika MeSH

• MeSH
• Bayesova věta MeSH
• interpretace statistických dat MeSH
• pravděpodobnost * MeSH
• soudní genetika MeSH
• Publikační typ
• rozhovory MeSH

AIM: A collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. METHODS: Eighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. RESULTS: Although the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. CONCLUSION: The laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized.

• MeSH
• DNA fingerprinting normy   MeSH
• DNA analýza   MeSH
• kosti a kostní tkáň chemie   MeSH
• lidé MeSH
• soudní genetika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Geografické názvy
• Česká republika MeSH

Human forensic STRs used for individual identification have been reported to have little power for inter-population analyses. Several methods have been developed which incorporate information on the spatial distribution of individuals to arrive at a description of the arrangement of diversity. We genotyped at 16 forensic STRs a large population sample obtained from many locations in Italy, Greece and Turkey, i.e. three countries crucial to the understanding of discontinuities at the European/Asian junction and the genetic legacy of ancient migrations, but seldom represented together in previous studies. Using spatial PCA on the full dataset, we detected patterns of population affinities in the area. Additionally, we devised objective criteria to reduce the overall complexity into reduced datasets. Independent spatially explicit methods applied to these latter datasets converged in showing that the extraction of information on long- to medium-range geographical trends and structuring from the overall diversity is possible. All analyses returned the picture of a background clinal variation, with regional discontinuities captured by each of the reduced datasets. Several aspects of our results are confirmed on external STR datasets and replicate those of genome-wide SNP typings. High levels of gene flow were inferred within the main continental areas by coalescent simulations. These results are promising from a microevolutionary perspective, in view of the fast pace at which forensic data are being accumulated for many locales. It is foreseeable that this will allow the exploitation of an invaluable genotypic resource, assembled for other (forensic) purposes, to clarify important aspects in the formation of local gene pools.

• MeSH
• genetická variace genetika   MeSH
• genotyp MeSH
• lidé MeSH
• mikrosatelitní repetice genetika   MeSH
• modely genetické * MeSH
• populační genetika * MeSH
• soudní genetika * MeSH
• zeměpis MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Středomoří MeSH

Stanovení doby úmrtí, resp. postmortálního intervalu (PMI), je jednou z nejproblematičtějších otázek ve forenzní praxi. Přesné určení PMI stále zůstává velmi komplikované i pro zkušené soudní lékaře. Přesný odhad PMI vyžaduje vyhodnocení takových parametrů, které se mění konstantně od doby úmrtí, nezávisle však na okolních vlivech. Dle současných výzkumů na poli molekulární biologie se zdá, že této definici bude odpovídat post mortem degradace nukleových kyselin (a to jak DNA, tak i RNA).

Estimation of time since death, i.e. the post-mortem interval (PMI), is one of the most problematic issues in forensic practice. Accurate determination of the PMI still remains very complicated task even for an experienced forensic pathologist. Physical changes including algor, livor and rigor mortis can be observed already during the first hours after death of an individual. Unfortunately, the estimation of PMI on the basis of these changes is often burdened with a certain degree of inaccuracy, which is caused by the temperature of surrounding environment, constitution of the body, cause of the death, location of the body, drug abuse etc. Accurate PMI estimation requires assessment of such parameters, which change constantly from the moment of death, but independently on ambient factors. According to current research in the field of molecular biology, it appears that a post-mortem degradation of nucleic acids (both DNA and RNA) will correspond to this definition.

• Klíčová slova
• postmortální interval, tafonomie,
• MeSH
• časové faktory * MeSH
• DNA genetika   MeSH
• lidé MeSH
• pitva MeSH
• posmrtné změny * MeSH
• RNA genetika   MeSH
• soudní genetika * metody   MeSH
• Check Tag
• lidé MeSH

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.

• MeSH
• amelogenin genetika   MeSH
• biochemická analýza krve MeSH
• DNA fingerprinting * MeSH
• genetické markery MeSH
• haplotypy MeSH
• laboratoře normy   MeSH
• lidé MeSH
• lidské chromozomy X * MeSH
• lidský chromozom Y * MeSH
• mikrosatelitní repetice * MeSH
• mitochondriální DNA genetika   MeSH
• sliny chemie   MeSH
• soudní genetika MeSH
• sperma chemie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH

Přístupy molekulární genetiky dovolují izolovat nukleové kyseliny ze vzorků (DNA i RNA současně) a analýzu těchto molekul využít nejen pro individuální identifikaci původců stop, ale také pro stanovení původu tělní tekutiny, která sama či ve směsi tvoří příslušnou biologickou stopu. Takto získané údaje mohou výrazně přispět ke zvýšení procesní hodnoty stopy. Molekulárně genetické metody zde diskutované nabízejí v porovnání s široce využívaným spektrem chemických a imunologických testů nové možnosti – je možné jimi analyzovat všechny typy biologických tekutin a jejich směsí, mají konfirmační charakter. Klíčová slova: tělní tekutiny– mRNA – mikroRNA – qPCR – metylace promotorů – mikrobiální DNA

Recent methodological approaches of molecular genetics allow isolation of nucleic acids (DNA and RNA) from negligible forensic samples. Analysis of these molecules may be used not only for individual identification based on DNA profiling but also for the detection of origin of the body fluid which (alone or in mixture with other body fluids) forms the examined biological trace. Such an examination can contribute to the evaluation of procedural, technical and tactical value of the trace. Molecular genetic approaches discussed in the review offer new possibilities in comparison with traditional spectrum of chemical, immunological and spectroscopic tests especially with regard to the interpretation of mixtures of biological fluids and to the confirmatory character of the tests. Approaches based on reverse transcription of tissue specific mRNA and their subsequent polymerase chain reaction (PCR) and fragmentation analysis are applicable on samples containing minimal amounts of biological material. Methods for body fluid discrimination based on examination of microRNA in samples provided so far confusing results therefore further development in this field is needed. The examination of tissue specific methylation of nucleotides in selected gene sequences seems to represent a promising enrichment of the methodological spectrum. The detection of DNA sequences of tissue related bacteria has been established and it provides satisfactory results mainly in combination with above mentioned methodological approaches. Keywords: body fluids – mRNA – microRNA – qPCR– promotermethylation – microbial DNA

• Klíčová slova
• metylace promotorů, mikrobiální DNA,
• MeSH
• bakteriální geny MeSH
• bakteriální RNA genetika   MeSH
• genetické markery MeSH
• krev MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• menstruace MeSH
• messenger RNA * MeSH
• metylace DNA genetika   MeSH
• mikro RNA MeSH
• moč chemie   MeSH
• pot chemie   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• sliny chemie   MeSH
• soudní genetika * metody   MeSH
• sperma chemie   MeSH
• stanovení celkové genové exprese MeSH
• tekutiny a sekrety tělesné * MeSH
• vagina chemie   mikrobiologie   sekrece   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH