International Society Of Forensic Genetics Research Articles

Introducing the New Editorial Team

Introducing the New Editorial Team

A Ge.F.I. – ISFG European collaborative study on DNA identification of Cannabis sativa samples using a 13-locus multiplex STR method

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists – International Society of Forensic Genetics (Ge.F.I. – ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of −1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

On the 65th birthday of Pavel Leonidovich Ivanov

Doctor of Biological Sciences, Professor Pavel Leonidovich Ivanov celebrated his 65th birthday on July 27, 2020. He is known as an internationally recognized scientist, an outstanding forensic geneticist, renowned both in Russia and in other countries, Deputy Director for High-Tech Research of the Russian Center for Forensic Science of the Ministry of Health of the Russian Federation, an expert of the Russian Academy of Sciences, a member of the International Society of Forensic Genetics and the European Network of Forensic Science Institutes (ENFSI), a full member of the World Academy of Science for Complex Safety, and an author of more than 300 scientific papers.

Recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics on forensic Y chromosome typing.

Y chromosome typing has been performed in forensic genetic practice for more than 20 years. The latest recommendations of the DNA Commission of the International Society of Forensic Genetics (ISFG) concerning the application of Y-chromosomal markers in forensic genetics were published in 2006. The aim of this report is to recapitulate, systematise and supplement existing recommendations on the forensic analysis of polymorphism of the Y chromosome with standards already implemented in practice, new capabilities linked to the development of research techniques as well as current solutions used in statistical analysis. The recommendations have been adapted specifically to aspects related to the preparation of expert opinions in the field of forensic genetics in Poland. The Polish Speaking Working Group of the ISFG believes that the presented guidelines should become a standard implemented by all Polish laboratories performing Y chromosome typing for forensic purposes.

Evaluation of 19 short tandem repeat markers for individualization of Papaver somniferum

Papaver somniferum, commonly known as opium poppy, is the source of natural opiates, which are used as analgesics or as precursors in the creation of semi-synthetic opioids such as heroin. An increase in opioid addiction in the United States has resulted in high rates of illicit opioid use and overdoses. It has recently been shown that P. somniferum DNA suitable for genetic analysis can be recovered from heroin samples. The development of a comprehensive genetic individualization tool for opium poppy could serve to link cases and strengthen programs such as the Drug Enforcement Administration’s (DEA) Heroin Signature Program, which seeks to combat rising opioid use.The purpose of this study was to develop a quantitative real-time PCR (qPCR) method for the quantification of opium poppy DNA, compare three commercial DNA extraction kits for their ability to isolate DNA from poppy seeds, and evaluate nineteen opium poppy short tandem repeat (STR) markers for their use in a forensic identification panel. Such a panel could be used for individualizing samples and determining the geographic origin in heroin or poppy seed tea cases. The qPCR method was proven to be reproducible and reliable, specific for P. somniferum, and sensitive enough for forensic case-type samples. Of the three kits tested, the nexttec™ one-step DNA Isolation Kit for Plants was the optimal method and facilitated rapid extraction of DNA from poppy seeds. The majority of evaluated STR primer sets were unreliable or had low discriminatory power, limiting their use for individualization of poppy samples. A six-locus STR multiplex was developed and evaluated according to Scientific Working Group on DNA Analysis Methods (SWGDAM) and International Society of Forensic Genetics (ISFG) guidelines, including the use of a sequenced allelic ladder. The multiplex was found to have low discriminatory power, with greater than two-thirds of samples analyzed having just two different genotypes. The multiplex was determined to be unsuitable for individualization; however, a genotype map was developed as a proof of concept that these markers may be useful for determining the biogeographical origin of samples. Searching the poppy genome for new STR markers and developing new primer sets may be necessary for the creation of a powerful genetic tool for the individualization of P. somniferum.

A large scale study on the characterisation of drop-in within a DNA laboratory

Forensic DNA analysis has become increasingly sensitive in recent years with the advent of improved PCR chemistries and highly sensitive CE instruments. This has allowed the detection of STR alleles from single copies of template DNA. As a consequence of this increased sensitivity and increases in the number of loci tested; understanding and characterising drop-in events become of greater importance. Drop-in is defined by the DNA commission of the International Society of Forensic Genetics as; one or two alleles that “come from different individuals” [1]. Many models have been developed to describe drop-in that can be applied to a continuous probability. However, no large study of drop-in has been published within the literature.

STRait Razor v2s: Advancing sequence-based STR allele reporting and beyond to other marker systems

STRait Razor has provided the forensic community a free-to-use, open-source tool for short tandem repeat (STR) analysis of massively parallel sequencing (MPS) data. STRait Razor v2s (SRv2s) allows users to capture physically phased haplotypes within the full amplicon of both commercial (ForenSeq) and “early access” panels (PowerSeq, Mixture ID). STRait Razor v2s may be run in batch mode to facilitate population-level analysis and is supported by all Unix distributions (including MAC OS). Data are reported in tables in string (haplotype), length-based (e.g., vWA allele 14), and International Society of Forensic Genetics (ISFG)-recommended (vWA [CE 14]-GRCh38-chr12:5983950-5984049 (TAGA)10 (CAGA)3 TAGA) formats. STRait Razor v2s currently contains a database of ∼2500 unique sequences. This database is used by SRv2s to match strings to the appropriate allele in ISFG-recommended format. In addition to STRs, SRv2s has configuration files necessary to capture and report haplotypes from all marker types included in these multiplexes (e.g., SNPs, InDels, and microhaplotypes). To facilitate mixture interpretation, data may be displayed from all markers in a format similar to that of electropherograms displayed by traditional forensic software. The download package for SRv2s may be found at https://www.unthsc.edu/graduate-school-of-biomedical-sciences/molecular-and-medical-genetics/laboratory-faculty-and-staff/strait-razor.

Collaborative EDNAP exercise on the IrisPlex system for DNA-based prediction of human eye colour.

The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on FTA cards by the organising laboratory together with eye colour phenotypes; 99.4% of the genotypes were correctly reported and 99% of the eye colour phenotypes were correctly predicted. Task 2 involved the assessment of 5 DNA samples extracted by the host laboratory from simulated casework samples, artificially degraded, and provided to the participants in varying DNA concentrations. For this task, 98.7% of the genotypes were correctly determined and 96.2% of eye colour phenotypes were correctly inferred. For Tasks 1 and 2 together, 99.2% (1875) of the 1890 genotypes were correctly generated and of the 15 (0.8%) incorrect genotype calls, only 2 (0.1%) resulted in incorrect eye colour phenotypes. The voluntary Task 3 involved participants choosing their own test subjects for IrisPlex genotyping and eye colour phenotype inference, while eye photographs were provided to the organising laboratory and judged; 96% of the eye colour phenotypes were inferred correctly across 100 samples and 19 laboratories. The high success rates in genotyping and eye colour phenotyping clearly demonstrate the reproducibility and the robustness of the IrisPlex assay as well as the accuracy of the IrisPlex model to predict blue and brown eye colour from DNA. Additionally, this study demonstrates the ease with which the IrisPlex system is implementable and applicable across forensic laboratories around the world with varying pre-existing experiences.

Validation of two canine STR multiplex-assays following the ISFG recommendations for non-human DNA analysis

To gain general acceptance forensic DNA testing in animals needs to improve standardization of analysis methods and data interpretation. Recently, the International Society of Forensic Genetics (ISFG) took particular care of this topic by publishing recommendations for forensic non-human DNA analysis following the successful example of human DNA analysis in order to provide a basis for harmonization of the still existing inter-laboratory variability. By following these recommendations we demonstrate the performance of two short tandem repeat (STR) multiplexes for forensic identity testing of canine biological material. Thirteen STRs and two sex-specific markers were selected and validated according to the ISFG guidelines. Population genetic parameters were calculated based on 295 dog samples collected in Austria (124) and Germany (171). A repeat-based nomenclature of the mainly tetrameric STRs and corresponding allelic ladders are presented. All 146 different alleles included in the ladders were sequenced for correct allele calling. Additionally, a canine cell line (DH82-D3167) was evaluated as standard reference material.

DNA commission of the International Society of Forensic Genetics: Recommendations on the evaluation of STR typing results that may include drop-out and/or drop-in using probabilistic methods

DNA profiling of biological material from scenes of crimes is often complicated because the amount of DNA is limited and the quality of the DNA may be compromised. Furthermore, the sensitivity of STR typing kits has been continuously improved to detect low level DNA traces. This may lead to (1) partial DNA profiles and (2) detection of additional alleles. There are two key phenomena to consider: allelic or locus 'drop-out', i.e. 'missing' alleles at one or more genetic loci, while 'drop-in' may explain alleles in the DNA profile that are additional to the assumed main contributor(s). The drop-in phenomenon is restricted to 1 or 2 alleles per profile. If multiple alleles are observed at more than two loci then these are considered as alleles from an extra contributor and analysis can proceed as a mixture of two or more contributors. Here, we give recommendations on how to estimate probabilities considering drop-out, Pr(D), and drop-in, Pr(C). For reasons of clarity, we have deliberately restricted the current recommendations considering drop-out and/or drop-in at only one locus. Furthermore, we offer recommendations on how to use Pr(D) and Pr(C) with the likelihood ratio principles that are generally recommended by the International Society of Forensic Genetics (ISFG) as measure of the weight of the evidence in forensic genetics. Examples of calculations are included. An Excel spreadsheet is provided so that scientists and laboratories may explore the models and input their own data.

A proposal for standardization in forensic bovine DNA typing: allele nomenclature of 16 cattle‐specific short tandem repeat loci

In this study, a proposal is presented for the allele nomenclature of 16 polymorphic short tandem repeat (STR) loci (BM1824, BM2113, ETH10, ETH225, INRA023, SPS115, TGLA122, TGLA126, TGLA227, ETH3, TGLA53, BM1818, CSRM60, CSSM66, HAUT27 and ILSTS006) for bovine genotyping (Bos taurus). The nomenclature is based on sequence data of the polymorphic region(s) of the STR loci as recommended by the DNA commission of the International Society of Forensic Genetics for human DNA typing. To cover commonly and rarely occurring alleles, a selection of animals homozygous for the alleles at these STR loci were analysed and subjected to sequence studies. The alleles of the STR loci consisted either of simple or compound dinucleotide repeat patterns. Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats including all variable regions within the amplified fragment. The set of 16 STR markers should be propagated for the use in all bovine applications including forensic analysis.

DNA commission of the International Society of Forensic Genetics: Recommendations on the interpretation of mixtures

The DNA commission of the International Society of Forensic Genetics (ISFG) was convened at the 21st congress of the International Society for Forensic Genetics held between 13 and 17 September in the Azores, Portugal. The purpose of the group was to agree on guidelines to encourage best practice that can be universally applied to assist with mixture interpretation. In addition the commission was tasked to provide guidance on low copy number (LCN) reporting. Our discussions have highlighted a significant need for continuing education and research into this area. We have attempted to present a consensus from experts but to be practical we do not claim to have conveyed a clear vision in every respect in this difficult subject. For this reason, we propose to allow a period of time for feedback and reflection by the scientific community. Then the DNA commission will meet again to consider further recommendations.

Results of the 2005 paternity testing workshop of the English Speaking Working Group of the International Society of Forensic Genetics

We present the results of the 2005 paternity testing workshop of the English Speaking Working Group of the International Society of Forensic Genetics. Blood samples from an alleged father, two children and a mother were sent to the participating laboratories. The participants were encouraged to treat the case as a paternity case, and also to consider other family relationships. A total of 62 laboratories reported results. The laboratories used a total of 42 autosomal STRs/PCR-investigated VNTRs, 22 Y-chromosomal STRs, 8 X-chromosomal STRs and 8 VNTR systems investigated with RFLP. The rate of typing and reporting errors was 0.3%. The results from a paper challenge showed that some of the rare events were treated differently in the participating laboratories.

DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. A previous recommendation published in 2001 has already addressed Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). Since then, the use of Y-STRs has become very popular, and numerous new loci have been introduced. The current recommendations address important aspects to clarify problems regarding the nomenclature, the definition of loci and alleles, population genetics and reporting methods

DNA Commission of the International Society of Forensic Genetics (ISFG): An update of the recommendations on the use of Y-STRs in forensic analysis

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. A previous recommendation published in 2001 has already addressed Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). Since then, the use of Y-STRs has become very popular, and a numerous new loci have been introduced. The current recommendations address important aspects to clarify problems regarding the nomenclature, the definition of loci and alleles, population genetics and reporting methods.

Polymorphism Data of Two STR Loci DYS632 and DYS634 in a Chinese Han Population

Blood samples were collected from 100 unrelated healthy males of the Chinese Han ethnic group in Chengdu of China. DNA was extracted using the Chelex method (1). The allelic variation at the two Y-STR loci named as DYS632 and DYS634 were analyzed by PCR amplification system. PCR amplification conditions can be accessed at: http://www.legalmed.org/dna/DYS632.htm. The volume of PCR reaction for each locus was 37.5 μL. The PCR products were analyzed by horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system and visualized by silver staining (2). Alleles were designated according to the recommendation of the International Society of Forensic Genetics (3). The gene diversity, the haplotype diversity, and the standard errors of diversity were calculated in accordance with Hou’s method (4). The complete data can be accessed at: http://www.legalmed. org/dna/DYS632.htm.

Results of the 2003 paternity testing workshop of the English Speaking Working Group of the International Society of Forensic Genetics

We present the results of the 2003 paternity testing workshop of the English Speaking Working Group (ESWG) of the International Society of Forensic Genetics (ISFG). The scenario was an alleged father, one child and the mother. All 51 participating laboratories drew the correct conclusion. The laboratories used a total of 30 autosomal and 17 Y-chromosomal PCR-based STR systems and 7 RFLP-based VNTR systems. The percentage of typing errors was 0.12% for the autosomal systems and 2% for the Y-chromosomal systems. The results from a paper challenge showed that occurrences of rare events such as mutations and possible silent alleles were treated differently among the participating laboratories. Participating laboratories reported mutational events observed in STR systems. Considerable differences between mutation rates in STR loci were reported. There were marked differences between paternal and maternal mutation rates as well as between single-step and multiple-step mutation rates.

Y-Chromosome STR Haplotypes in a Han Ethnic Group of Chinese Population

Abstract Blood samples were obtained from 120 healthy unrelated males from the Han ethnic group in Chengdu of China. The DNA was extracted by using Chelex 100 protocol as described by Walsh et al. (1). The allelic variation at the three Y-STR loci, named as DYS447, DYS449 and DYS450, were analyzed by PCR amplification system. Each PCR reaction was performed in a 37.5 µL containing 2-10 ng DNA, 1 X Taq buffer, 1.5 mM MgCl2, 200 RM each dNTP (Pharmacia Biotech, Sweden), 1.5 U Taq polymerase (NEB, UK), 0.3 RM each primer in a Perkin-Elmer 9600 thermocycler (ABI, Foster City, CA). The PCR products were analyzed by horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system and visualized by silver staining (2). Alleles were designated according to recommendation of the International Society of Forensic Genetics (3). The gene diversities, the haplotypes diversity and the standard errors of diversity were calculated in accordance with Hou's method (4).

Allele Frequencies of Two Y-STRs in a Chinese Population

Abstract A total of 74 blood samples were collected from unrelated males of Han ethnic group in Chongqing of China. DNA was extracted by utilizing Chelex method (1). Each PCR reaction contained 2-10 ng DNA, 1 X Taq buffer, 1.5 mM MgCl2, 200 RM each dNTP (Pharmacia Biotech, Sweden), 1.5 U Taq polymerase (NEB, UK), 0.3 RM each primer. PCR amplifications were performed in a GeneAmp PCR System 9600 (Perkin-Elmer, Foster City, CA),with denaturing for 2 min at 94°C, followed by 30 cycles of 94°C for 50 s, 58°C for 50 s and 72°C for 30 s. PCR products were analyzed by horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system, gels were silver stained (2). Alleles were designated according to recommendation of the International Society of Forensic Genetics (3). The gene diversities, the haplotypes diversity, and the standard errors of diversity were calculated in accordance with Hou's method (4).

Distributions of Allelic Frequencies and Haplotypes of Two New Y-STR Loci in a Chinese Han Population

Abstract Blood samples were collected from 109 unrelated males of Han ethnic group in Chengdu of China. DNA was extracted using Chelex method (1). The volume of PCR reaction for each locus was 37.5 µL, which contained 2-10 ng human genome, 1 X Taq buffer, 1.5 mM MgCl2, 1.6 . g/mL BSA, 200 RM each dNTP (Pharmacia Biotech, Sweden), 1.5 U Taq polymerase (Promega Corporation, Madison, WI), 0.3 RM each primer. PCR amplifications were carried out in a GeneAmp PCR System 9600 (Perkin-Elmer, Foster City, CA) with pre-denaturing for 3 min at 94°C, 36 cycles of denaturing for 30 s at 94°C, annealing for 60 s at 61°C and extension for 30 s at 72°C. The amplicons were analyzed by horizontal nondenaturing polyacrylamide gel electrophoresis with discontinuous buffer system and visualized by silver staining (2). Alleles were designated according to recommendation of the DNA commission of the International Society of Forensic Genetics (3). Data of population genetics and forensic science were analyzed according to Hou's method (4).