Recommendations of the DNA Commission of the International Society for Forensic Genetics (ISFG) on short tandem repeat sequence nomenclature.

The DNA Commission of the International Society for Forensic Genetics (ISFG) has developed a set of nomenclature recommendations for short tandem repeat (STR) sequences. These recommendations follow the 2016 considerations of the DNA Commission of the ISFG, incorporating the knowledge gained through research and population studies in the intervening years. While maintaining a focus on backward compatibility with the CE data that currently populate national DNA databases, this report also looks to the future with the establishment of recommended minimum sequence reporting ranges to facilitate interlaboratory comparisons, automated solutions for sequence-based allele designations, a suite of resources to support bioinformatic development, guidance for characterizing new STR loci, and considerations for incorporating STR sequences and other new markers into investigative databases.

Recent advances in forensic biology and forensic DNA typing: INTERPOL review 2019-2022.

This review paper covers the forensic-relevant literature in biological sciences from 2019 to 2022 as a part of the 20th INTERPOL International Forensic Science Managers Symposium. Topics reviewed include rapid DNA testing, using law enforcement DNA databases plus investigative genetic genealogy DNA databases along with privacy/ethical issues, forensic biology and body fluid identification, DNA extraction and typing methods, mixture interpretation involving probabilistic genotyping software (PGS), DNA transfer and activity-level evaluations, next-generation sequencing (NGS), DNA phenotyping, lineage markers (Y-chromosome, mitochondrial DNA, X-chromosome), new markers and approaches (microhaplotypes, proteomics, and microbial DNA), kinship analysis and human identification with disaster victim identification (DVI), and non-human DNA testing including wildlife forensics. Available books and review articles are summarized as well as 70 guidance documents to assist in quality control that were published in the past three years by various groups within the United States and around the world.

DNA commission of the International Society of Forensic Genetics (ISFG): Recommendations on the interpretation of Y-STR results in forensic analysis.

Forensic genetic laboratories perform a large amount of STR analyses of the Y chromosome, in particular to analyze the male part of complex DNA mixtures. However, the statistical interpretation of evidence retrieved from Y-STR haplotypes is challenging. Due to the uni-parental inheritance mode, Y-STR loci are connected to each other and thus haplotypes show patterns of relationship on the familial and population level. This precludes the treatment of Y-STR loci as independently inherited variables and the application of the product rule. Instead, the dependency structure of Y-STRs needs to be included in the haplotype frequency estimation process affecting also the current paradigm of a random match probability that is in the autosomal case approximated by the population frequency assuming unrelatedness of sampled individuals. Information on the degree of paternal relatedness in the suspect population as well as on the familial network is however needed to interpret Y-chromosomal results in the best possible way. The previous recommendations of the DNA commission of the ISFG on the use of Y-STRs in forensic analysis published more than a decade ago [1] cover the interpretation issue only marginally. The current recommendations address a number of topics (frequency estimators, databases, metapopulations, LR formulation, triage, rapidly mutating Y-STRs) with relevance for the Y-STR statistics and recommend a decision-based procedure, which takes into account legal requirements as well as availability of population data and statistical methods.

Ethical publication of research on genetics and genomics of biological material: guidelines and recommendations.

Forensic Science International: Genetics and Forensic Science International: Reports communicate research on a variety of biological materials using genetics and genomic methods. Numerous guidelines have been produced to secure standardization and quality of results of scientific investigations. Yet, no specific guidelines have been produced for the ethical acquisition of such data. These guidelines summarize universally adopted principles for conducting ethical research on biological materials, and provide details of the general procedures for conducting ethical research on materials of human, animal, plant and environmental origin. Finally, the minimal ethics requirements for submission of research material are presented.

DNA commission of the International society for forensic genetics: Assessing the value of forensic biological evidence - Guidelines highlighting the importance of propositions. Part II: Evaluation of biological traces considering activity level propositions.

The value of the evidence depends critically on propositions. In the second of two papers intended to provide advice to the community on difficult aspects of evaluation and the formulation of propositions, we focus primarily on activity level propositions. This helps the court address the question of "How did an individual's cell material get there?". In order to do this, we expand the framework outlined in the first companion paper. First, it is important not to conflate results and propositions. Statements given activity level propositions aim to help address issues of indirect vs direct transfer, and the time of the activity, but it is important to avoid use of the word 'transfer' in propositions. This is because propositions are assessed by the Court, but DNA transfer is a factor that scientists need to take into account for the interpretation of their results. Suitable activity level propositions are ideally set before knowledge of the results and address issues like: X stabbed Y vs. an unknown person stabbed Y but X met Y the day before. The scientist assigns the probability of the evidence, if each of the alternate propositions is true, to derive a likelihood ratio. To do this, the scientist asks: a) "what are the expectations if each of the propositions is true?" b) "What data are available to assist in the evaluation of the results given the propositions?" When presenting evidence, scientists work within the hierarchy of propositions framework. The value of evidence calculated for a DNA profile cannot be carried over to higher levels in the hierarchy - the calculations given sub-source, source and activity level propositions are all separate. A number of examples are provided to illustrate the principles espoused, and the criteria that such assessments should meet. Ideally in order to assign probabilities, the analyst should have/collect data that are relevant to the case in question. These data must be relevant to the case at hand and we encourage further research and collection of data to form knowledge bases. Bayesian Networks are extremely useful to help us think about a problem, because they force us to consider all relevant possibilities in a logical way. An example is provided.

Interpol review of forensic biology and forensic DNA typing 2016-2019.

This review paper covers the forensic-relevant literature in biological sciences from 2016 to 2019 as a part of the 19th Interpol International Forensic Science Managers Symposium. The review papers are also available at the Interpol website at: https://www.interpol.int/content/download/14458/file/Interpol%20Review%20Papers%202019.pdf.

NIST interlaboratory studies involving DNA mixtures (MIX05 and MIX13): Variation observed and lessons learned.

Interlaboratory studies are a type of collaborative exercise in which many laboratories are presented with the same set of data to interpret, and the results they produce are examined to get a "big picture" view of the effectiveness and accuracy of analytical protocols used across participating laboratories. In 2005 and again in 2013, the Applied Genetics Group of the National Institute of Standards and Technology (NIST) conducted interlaboratory studies involving DNA mixture interpretation. In the 2005 NIST MIX05 study, 69 laboratories interpreted data in the form of electropherograms of two-person DNA mixtures representing four different mock sexual assault cases with different contributor ratios. In the 2013 NIST MIX13 study,108 laboratories interpreted electropherogram data for five different case scenarios involving two, three, or four contributors, with some of the contributors potentially related. This paper describes the design of these studies, the variations observed among laboratory results, and lessons learned.

DNA commission of the International society for forensic genetics: Assessing the value of forensic biological evidence - Guidelines highlighting the importance of propositions: Part I: evaluation of DNA profiling comparisons given (sub-) source propositions.

The interpretation of evidence continues to be one of the biggest challenges facing the forensic community. This is the first of two papers intended to provide advice on difficult aspects of evaluation and in particular on the formulation of propositions. The scientist has a dual role: investigator (crime-focused), where often there is no suspect available and a database search may be required; evaluator (suspect-focused), where the strength of evidence is assessed in the context of the case. In investigative mode, generally the aim is to produce leads regarding the source of the DNA. Sub-source level propositions will be adequate to help identify potential suspects who can be further investigated by the authorities. Once in evaluative mode, given the defence version of events of the person of interest, it may become necessary to consider alternatives that go beyond the source of the DNA (i.e., to consider activity level propositions). In the evaluation phase, it is crucial that formulation of propositions allows the assessment of all the results that will help with the issue at hand. Propositions should therefore be precise (indication of the number of contributors, information on the relevant population etc.), be about causes, not effects (e.g. a 'matching' DNA profile) and to avoid bias, must not be findings-led. This means that ideally, propositions should be decided based on the case information and before the results of the comparisons are known. This paper primarily reflects upon what has been coined as "sub-source level propositions". These are restricted to the evaluation of the DNA profiles themselves, and help answer the issue regarding the source of the DNA. It is to be emphasised that likelihood ratios given sub-source level propositions cannot be carried over to a different level - for example, activity level propositions, where the DNA evidence is put into the context of the alleged activities. This would be highly misleading and could give rise to miscarriages of justice; this will be discussed in a second paper. The value of forensic results depends not only on propositions, but also on the type of results (e.g. allelic designations, peak heights, replicates) and upon the model used: it is therefore important to discuss these aspects. Finally, since communication is key to help understanding by courts, we will explore how to convey the value of the results and explain the importance of avoiding the practice of transposing the conditional.

DNA Commission of the International Society for Forensic Genetics (ISFG): Guidelines on the use of X-STRs in kinship analysis.

Forensic genetic laboratories perform an increasing amount of genetic analyses of the X chromosome, in particular to solve complex cases of kinship analysis. For some biological relationships X-chromosomal markers can be more informative than autosomal markers, and there are a large number of markers, methods and databases that have been described for forensic use. Due to their particular mode of inheritance, and their physical location on a single chromosome, some specific considerations are required when estimating the weight of evidence for X-chromosomal marker DNA data. The DNA Commission of the International Society for Forensic Genetics (ISFG) hereby presents guidelines and recommendations for the use of X-chromosomal markers in kinship analysis with a special focus on the biostatistical evaluation. Linkage and linkage disequilibrium (association of alleles) are of special importance for such evaluations and these concepts and the implications for likelihood calculations are described in more detail. Furthermore it is important to use appropriate computer software that accounts for linkage and linkage disequilibrium among loci, as well as for mutations. Even though some software exist, there is still a need for further improvement of dedicated software.

Evaluation of forensic DNA mixture evidence: protocol for evaluation, interpretation, and statistical calculations using the combined probability of inclusion.

BACKGROUND: The evaluation and interpretation of forensic DNA mixture evidence faces greater interpretational challenges due to increasingly complex mixture evidence. Such challenges include: casework involving low quantity or degraded evidence leading to allele and locus dropout; allele sharing of contributors leading to allele stacking; and differentiation of PCR stutter artifacts from true alleles. There is variation in statistical approaches used to evaluate the strength of the evidence when inclusion of a specific known individual(s) is determined, and the approaches used must be supportable. There are concerns that methods utilized for interpretation of complex forensic DNA mixtures may not be implemented properly in some casework. Similar questions are being raised in a number of U.S. jurisdictions, leading to some confusion about mixture interpretation for current and previous casework. RESULTS: Key elements necessary for the interpretation and statistical evaluation of forensic DNA mixtures are described. Given the most common method for statistical evaluation of DNA mixtures in many parts of the world, including the USA, is the Combined Probability of Inclusion/Exclusion (CPI/CPE). Exposition and elucidation of this method and a protocol for use is the focus of this article. Formulae and other supporting materials are provided. CONCLUSIONS: Guidance and details of a DNA mixture interpretation protocol is provided for application of the CPI/CPE method in the analysis of more complex forensic DNA mixtures. This description, in turn, should help reduce the variability of interpretation with application of this methodology and thereby improve the quality of DNA mixture interpretation throughout the forensic community.

Recommendations of the DNA Commission of the International Society for Forensic Genetics (ISFG) on quality control of autosomal Short Tandem Repeat allele frequency databasing (STRidER).

The statistical evaluation of autosomal Short Tandem Repeat (STR) genotypes is based on allele frequencies. These are empirically determined from sets of randomly selected human samples, compiled into STR databases that have been established in the course of population genetic studies. There is currently no agreed procedure of performing quality control of STR allele frequency databases, and the reliability and accuracy of the data are largely based on the responsibility of the individual contributing research groups. It has been demonstrated with databases of haploid markers (EMPOP for mitochondrial mtDNA, and YHRD for Y-chromosomal loci) that centralized quality control and data curation is essential to minimize error. The concepts employed for quality control involve software-aided likelihood-of-genotype, phylogenetic, and population genetic checks that allow the researchers to compare novel data to established datasets and, thus, maintain the high quality required in forensic genetics. Here, we present STRidER (http://strider.online), a publicly available, centrally curated online allele frequency database and quality control platform for autosomal STRs. STRidER expands on the previously established ENFSI DNA WG STRbASE and applies standard concepts established for haploid and autosomal markers as well as novel tools to reduce error and increase the quality of autosomal STR data. The platform constitutes a significant improvement and innovation for the scientific community, offering autosomal STR data quality control and reliable STR genotype estimates.

Massively parallel sequencing of forensic STRs: Considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements.

The DNA Commission of the International Society for Forensic Genetics (ISFG) is reviewing factors that need to be considered ahead of the adoption by the forensic community of short tandem repeat (STR) genotyping by massively parallel sequencing (MPS) technologies. MPS produces sequence data that provide a precise description of the repeat allele structure of a STR marker and variants that may reside in the flanking areas of the repeat region. When a STR contains a complex arrangement of repeat motifs, the level of genetic polymorphism revealed by the sequence data can increase substantially. As repeat structures can be complex and include substitutions, insertions, deletions, variable tandem repeat arrangements of multiple nucleotide motifs, and flanking region SNPs, established capillary electrophoresis (CE) allele descriptions must be supplemented by a new system of STR allele nomenclature, which retains backward compatibility with the CE data that currently populate national DNA databases and that will continue to be produced for the coming years. Thus, there is a pressing need to produce a standardized framework for describing complex sequences that enable comparison with currently used repeat allele nomenclature derived from conventional CE systems. It is important to discern three levels of information in hierarchical order (i) the sequence, (ii) the alignment, and (iii) the nomenclature of STR sequence data. We propose a sequence (text) string format the minimal requirement of data storage that laboratories should follow when adopting MPS of STRs. We further discuss the variant annotation and sequence comparison framework necessary to maintain compatibility among established and future data. This system must be easy to use and interpret by the DNA specialist, based on a universally accessible genome assembly, and in place before the uptake of MPS by the general forensic community starts to generate sequence data on a large scale. While the established nomenclature for CE-based STR analysis will remain unchanged in the future, the nomenclature of sequence-based STR genotypes will need to follow updated rules and be generated by expert systems that translate MPS sequences to match CE conventions in order to guarantee compatibility between the different generations of STR data.

STR allele sequence variation: Current knowledge and future issues.

This article reviews what is currently known about short tandem repeat (STR) allelic sequence variation in and around the twenty-four loci most commonly used throughout the world to perform forensic DNA investigations. These STR loci include D1S1656, TPOX, D2S441, D2S1338, D3S1358, FGA, CSF1PO, D5S818, SE33, D6S1043, D7S820, D8S1179, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D18S51, D19S433, D21S11, Penta D, and D22S1045. All known reported variant alleles are compiled along with genomic information available from GenBank, dbSNP, and the 1000 Genomes Project. Supplementary files are included which provide annotated reference sequences for each STR locus, characterize genomic variation around the STR repeat region, and compare alleles present in currently available STR kit allelic ladders. Looking to the future, STR allele nomenclature options are discussed as they relate to next generation sequencing efforts underway.

U.S. initiatives to strengthen forensic science & international standards in forensic DNA.

A number of initiatives are underway in the United States in response to the 2009 critique of forensic science by a National Academy of Sciences committee. This article provides a broad review of activities including efforts of the White House National Science and Technology Council Subcommittee on Forensic Science and a partnership between the Department of Justice (DOJ) and the National Institute of Standards and Technology (NIST) to create the National Commission on Forensic Science and the Organization of Scientific Area Committees. These initiatives are seeking to improve policies and practices of forensic science. Efforts to fund research activities and aid technology transition and training in forensic science are also covered. The second portion of the article reviews standards in place or in development around the world for forensic DNA. Documentary standards are used to help define written procedures to perform testing. Physical standards serve as reference materials for calibration and traceability purposes when testing is performed. Both documentary and physical standards enable reliable data comparison, and standard data formats and common markers or testing regions are crucial for effective data sharing. Core DNA markers provide a common framework and currency for constructing DNA databases with compatible data. Recent developments in expanding core DNA markers in Europe and the United States are discussed.

The future of forensic DNA analysis.

The author's thoughts and opinions on where the field of forensic DNA testing is headed for the next decade are provided in the context of where the field has come over the past 30 years. Similar to the Olympic motto of 'faster, higher, stronger', forensic DNA protocols can be expected to become more rapid and sensitive and provide stronger investigative potential. New short tandem repeat (STR) loci have expanded the core set of genetic markers used for human identification in Europe and the USA. Rapid DNA testing is on the verge of enabling new applications. Next-generation sequencing has the potential to provide greater depth of coverage for information on STR alleles. Familial DNA searching has expanded capabilities of DNA databases in parts of the world where it is allowed. Challenges and opportunities that will impact the future of forensic DNA are explored including the need for education and training to improve interpretation of complex DNA profiles.