Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories.Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male).All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data.As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland.Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.     

Forensic typing of autosomal SNPs with a 29 SNP-multiplex—Results of a collaborative EDNAP exercise

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.     

STR analysis of artificially degraded DNA-results of a collaborative European exercise

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.     

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.     

Report of the blind trial of the Cetus Amplitype HLA DQ alpha forensic deoxyribonucleic acid (DNA) amplification and typing kit

The AmpliType HLA DQ alpha forensic DNA amplification and typing kit is designed for the qualitative analysis of the human leukocyte antigen (HLA) DQ alpha alleles present in deoxyribonucleic acid (DNA) extracted from forensic samples. The AmpliType kit is the first forensic DNA typing product based on the GeneAmp polymerase chain reaction (PCR) process. The kit was evaluated by five forensic science laboratories (test sites) to assess their ability to perform DNA typing using PCR on sample types typically encountered by forensic laboratories. None of the DNA-containing samples was mistyped. Of the 180 DNA-containing samples analyzed, results were reported for 178 (98.9%). Of the 178 samples with results, all were correctly typed. Two sites did not report a result for one sample each. Four of the five laboratories experienced no significant levels of contamination in the DNA-containing samples. At the one site with the highest number of DNA-containing samples with contamination, the typing results were not compromised. This site was able to correct the contamination problem through simple procedural changes and stricter attention to sterile technique. Blank controls were important to monitor contamination. In conclusion, the trial demonstrated that forensic science laboratories are capable of setting up a PCR-based DNA typing laboratory and successfully using the AmpliType HLA DQ alpha forensic DNA amplification and typing kit to analyze forensic samples.     

Results of collaborative study regarding the standardization of the Y-linked STR system DYS385 by the European DNA Profiling (EDNAP) group.

Y-chromosome linked short tandem repeat (STR) loci are inherited as a closely linked haplotype, which appears to remain stable in a given paternal lineage over many generations. In forensic cases, Y-linked STRs are particularly useful for the identification of human remains as well as in rape cases with mixed male/female stain samples. DYS385 is derived from tandemly duplicated segments of the Y chromosome thus giving rise to two fragments of variable length which do not behave like alleles but genotypes. The European DNA Profiling (EDNAP) group has carried out a collaborative exercise among 14 participating laboratories using DYS385 for typing of five unknown bloodstains and a control sample. Furthermore, population data from eight different European countries with samples sizes between 91 and 150 male individuals were collected. The results confirm previous observations that DYS385 is one of the most informative Y-linked STR loci. It could also be demonstrated that reproducible results can be obtained independently from the electrophoretic separation and detection methods used. Thus DYS385 may serve as a useful complementation to the routinely used autosomal STR systems in special cases.     

Evaluation of the catalytic decomposition of H2O2 through use of organo-metallic complexes--a potential link to the luminol presumptive blood test

Hair testing for drugs of abuse is performed in Lombardy by eleven analytical laboratories accredited for forensic purposes, the most frequent purposes being driving license regranting and workplace drug testing. Individuals undergoing hair testing for these purposes can choose the laboratory in which the analyses have to be carried out. The aim of our study was to perform an interlaboratory exercise in order to verify the level of standardization of hair testing for drugs of abuse in these accredited laboratories; nine out of the eleven laboratories participated in this exercise. Sixteen hair strands coming from different subjects were longitudinally divided in 3-4 aliquots and distributed to participating laboratories, which were requested to apply their routine methods. All the participants analyzed opiates (morphine and 6-acetylmorphine) and cocainics (cocaine and benzoylecgonine) while only six analyzed methadone and amphetamines (amphetamine, methamphetamine, MDMA, MDA and MDEA) and five Δ(9)-tetrahydrocannabinol (THC). The majority of the participants (seven labs) performed acidic hydrolysis to extract the drugs from the hair and analysis by GC-MS, while two labs used LC-MS/MS. Eight laboratories performed initial screening tests by Enzyme Multiplied Immunoassay Technique (EMIT), Enzyme-linked Immunosorbent Assay (ELISA) or Cloned Enzyme Donor Immunoassay (CEDIA). Results demonstrated a good qualitative performance for all the participants, since no false positive results were reported by any of them. Quantitative data were quite scattered, but less in samples with low concentrations of analytes than in those with higher concentrations. Results from this first regional interlaboratory exercise show that, on the one hand, individuals undergoing hair testing would have obtained the same qualitative results in any of the nine laboratories. On the other hand, the scatter in quantitative results could cause some inequalities if any interpretation of the data is required.     

A collaborative study of the EDNAP group regarding Y-chromosome binary polymorphism analysis

A collaborative study was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the performance of Y-chromosome binary polymorphism analysis in different European laboratories. Four blood samples were sent to the laboratories, to be analysed for 11 Y-chromosome single nucleotide polymorphisms (SNPs): SRY-1532, M40, M35, M213, M9, 92R7, M17, P25, M18, M153 and M167. All the labs were also asked to submit a population study including these markers. All participating laboratories reported the same results, indicating the reproducibility and robustness of Y-chromosome SNP typing. A total of 535 samples from six different European populations were also analysed. In Galicia (NW Spain) and Belgium, the most frequent haplogroup was R1b*(xR1b1,R1b3df). Haplogroup F*(xK) is one of the most frequent in Austria and Denmark, while the lowest frequency appear in Belgium. Haplogroup frequencies found in this collaborative study were compared with previously published European Y-chromosome haplogroup data.     

Results of the GEP-ISFG collaborative study on two Y-STRs tetraplexes: GEPY I (DYS461, GATA C4, DYS437 and DYS438) and GEPY II (DYS460, GATA A10, GATA H4 and DYS439)

A collaborative exercise was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) in order to evaluate the performance of two Y-chromosome STR PCR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The participating laboratories were asked to type three samples for the eight markers, using a specific amplification protocol. In addition, two control samples, with known haplotypes, were provided. The results obtained by the 13 different participating laboratories were identical, except for two laboratories that failed to type correctly the same two samples for GATA C4. By sequence analyses, two different GATA C4 allele structures were found. One control sample (allele 21) and two questioned samples (allele 22, correctly typed by all the laboratories, and allele 25) presented the following repeat structure: (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)n, but different from the one found for allele 26 in one sample included in this exercise, as well as in the second control sample (allele 23), namely (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)2(TGTA)2(TCTA)n. The collaborative exercise results proved that both Y-tetraplexes produce good amplification results, with the advantage of being efficiently typed using different separation and detection methodologies. However, since GATA C4 repeat presents a complex structure, with alleles differing in sequence structure, efficient denaturing conditions should be followed in order to avoid typing errors due to sizing problems.     

国外法庭科学DNA实验室的质量保证和质量控制现状

法庭科学DNA检测飞速发展和广泛应用的同时也面临巨大风险,实验室质量保证能力和质量控制手段的不足已开始影响到法庭科学DNA检测的证据地位。本文对国外法庭科学DNA实验室的有关情况进行初步分析,从中发掘有益的启示,为我国法庭科学DNA检测的改革和发展提供借鉴。     

Species identification in routine casework samples using the SPInDel kit

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identification by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 to 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95% of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.     

RNA/DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.     

Report on the third EDNAP collaborative STR exercise

This report describes an inter-laboratory exercise completed on behalf of the European DNA Profiling (EDNAP) group. The exercise is one in a series designed to identify STR loci which could be used for harmonisation between participating European forensic science laboratories. Participants were asked to identify the alleles present in five bloodstains at the STR loci HUMTHO1 and HUMVWFA31/A. Two of the stains were prepared from mixtures of two different blood samples. There were no special instructions and each laboratory was requested to use the methodology normally employed for crime case investigations. All participating laboratories achieved the same results for both loci. In addition, the laboratories were also requested to report the results obtained from any other loci which would normally be used in crime case investigations. A comparison of these results showed some inter-laboratory variation.     

Quantifiler Human DNA Quantification Kit (Applied Biosystems) as a screening kit for DNA profiling

This study investigates the connection between the results of DNA quantification with Quantifiler Human DNA Quantification Kit (AB) and DNA profiling. For this purpose the DNA concentration of 3.068 routine casework samples was determined and DNA profiling was carried out. For discussion, depending on the specific DNA concentration, the samples were divided into four groups (0–5, 5–10, 10–30 and more then 30 pg/μl DNA) and the obtained number of full or partial profiles and the negative typing results was listed. Moreover group 1 (0–5 pg/μl DNA) results were subdivided and analysed more precisely. Based on the amount of 4% positive typing results in group 1 we decided to analyse every sample with quantification results >0 pg/μl DNA. A real cut-off no longer exists. Only samples showing 0 pg/μl on each result were sorted out.     

Performance evaluation of two multiplexes used in fluorescent short tandem repeat DNA analysis

The performance of two commercial multiplex kits that together amplify the 13 core short tandem repeat (STR) loci currently in use by forensic laboratories and the U.S. national Combined DNA Indexing System (CODIS) were evaluated. The typing systems examined were AmpFlSTR Profiler Plus and AmpFlSTR COfiler (PE Applied Biosystems, Foster City, CA). Electrophoretic separation and detection of the fluorescent PCR products was achieved by capillary electrophoresis (CE) using an ABI Prism 310 Genetic Analyzer. The studies addressed the on-site validation of the instrument, the software, and each typing system. These studies included instrument sensitivity, resolution, precision, binning, peak height ratios, mixtures, stutter, and the amplification of non-probative and simulated forensic samples. Other additional developmental-type work is also reported herein, such as species specificity testing and amplification of environmentally insulted samples. Amplification conditions were found to be robust and the primer sets shown to be specific to human DNA. Stutter and peak height ratios fell within limits published by the manufacturer and other laboratories. The data demonstrate that the CE instrument can consistently resolve fragments differing in length by one base and that the +/-0.5 base bin used by the Genotyper software is acceptable for making accurate allele calls. Correct typing results were obtained from non-probative and simulated case samples, as well as samples exposed to outdoor environmental conditions. The results support the conclusion that DNA extracted from biological samples routinely encountered in the forensic laboratory can be reliably analyzed with AmpFlSTR Profiler Plus and COfiler using CE.     

Results from the NIST 2004 DNA Quantitation Study

For optimal DNA short tandem repeat (STR) typing results, the DNA concentration ([DNA]) of the sample must be accurately determined prior to the polymerase chain reaction (PCR) amplification step in the typing process. In early 2004, the National Institute of Standards and Technology (NIST) conducted an interlaboratory study to help assess the accuracy of DNA quantitation in forensic DNA laboratories. This study was designed with four primary purposes: (1) to examine concentration effects and to probe performance at the lower DNA concentration levels that are frequently seen in forensic casework; (2) to examine consistency with various methodologies across multiple laboratories; (3) to examine single versus multiple source samples; and (4) to study DNA stability over time and through shipping in two types of storage tubes. Eight DNA samples of [DNA] from 0.05 ng/microL to 1.5 ng/microL were distributed. A total of 287 independent data sets were returned from 80 participants. Results were reported for 19 different DNA quantitation methodologies. Approximately 65% of the data were obtained using traditional slot blot hybridization methods; 21% were obtained using newly available quantitative real-time PCR (Q-PCR) techniques. Information from this interlaboratory study is guiding development of a future NIST Standard Reference Material for Human DNA Quantitation, SRM 2372.     

Detection of a primer-binding site polymorphism for the STR locus D16S539 using the Powerplex 1.1 system and validation of a degenerate primer to correct for the polymorphism

Quality assurance samples submitted from the NCSBI as part of a contract with TBTG to outsource DNA Database samples showed unexpected discrepancies for the locus D16S539 when all other loci yielded identical results. Discrepancies observed included allele drop out and an imbalance in sister alleles with samples returned from TBTG. This led to a comprehensive review of the technical procedures used between the two laboratories to determine the cause of the discrepancies noted for the locus D16S539, since both laboratories were using the PowerPlex 1.1 typing kit from the Promega Corporation. The NCSBI and the TBTG utilize different extraction methods (organic extraction vs. FTA) and amplification conditions (AmpliTaq vs AmpliTaq Gold), respectively, so the exact cause of discrepancy observed was not immediately apparent. Experiments at the NCSBI associated the observed allele drop out and the imbalance of the sister alleles with the use of AmpliTaq Gold and a hot start procedure. Sequencing data revealed that a point mutation resides on the D16S539 primer-binding site that reaches polymorphic levels in African-American populations. This led to the development of a degenerate primer by the Promega Corporation to detect "missing" alleles when AmpliTaq Gold is used. The degenerate primer was then thoroughly tested to show its efficacy in detecting the "true" D16S539 profile when used.     

不同分型方法的STR分型差异

目的调查不同的STR分型系统之间分型的一致性。方法 10 0例不同个体的DNA样本分别用单位点聚丙烯酰胺凝胶银染法和PowerPlex16System试剂盒对 13个法医学常用STR位点进行基因分型 ,并比较两种不同分型系统间的分型结果。结果 1例样本在D8S1179位点出现了分型不一致的结果 :银染法的基因型为 12 / 14 ,而用PowerPlex16System试剂盒的分型则为 12 / 15。结论不同的STR分型系统可导致不同的基因分型     

人骨骼及牙齿DNA提取方法的比较和优化

目的人骨骼和牙齿DNA提取方法的比较和优化。方法收集18份不同个体的长骨、30颗磨牙和同一个体2根股骨、8颗磨牙。利用TissueLyser-Ⅱ组织破碎仪和PreFiler Express BTA^TM法医DNA提取试剂盒（BTA法）,应用Automate Express^TM自动化法医DNA提取系统提取DNA,进行STR分型,与脱钙法进行比较,并进行实验条件优化。结果用TissueLyser-Ⅱ结合BTA法,约2.5h即可完成骨骼和牙齿的DNA提取,分型成功率分别为94.4%和96.7%。与脱钙法比较,两种方法获得DNA质量浓度和检出率比较接近（P〈0.05）,但BTA法在操作过程方面更具优势。最佳样本量为100mg,消化时间为2h。结论采用TissueLyser-Ⅱ组织破碎仪结合BTA法对骨骼和牙齿进行DNA提取和分型检验,能满足实际检案的要求,可在法医学实践中选择使用。