An inter-laboratory study of DNA-based identity,parentage and species testing in animal forensic genetics

The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability.Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output.The International Society for Animal Genetics(ISAG)administered animal forensic comparison tests(AFCTs)in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification,parentage and species determination services.The AFCTs revealed that analyses of low DNA template concentrations(≤300 pg/μL)constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results.Moreover,a lack of familiarity with species testing protocols,interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results.Several laboratories showed improvement in their genotyping accuracy over time.However,the use of forensically validated standards,such as a standard forensic short tandem repeat(STR)kit,preferably with an allelic ladder,and stricter guidelines for STR typing,may have prevented some common issues from occurring,such as genotyping inaccuracies,missing data,elevated stutter products and loading errors.The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other.Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel’s proficiency in critical techniques such as low copy number(LCN)analysis and species testing.Although this is the first time that the ISAG has conducted comparison tests for forensic testing,findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.

Improving DNA Data Capacity:Forensic Parameters and Genetic Structure Analysis of Jinjiang Han Population with the Microreader^(TM) Y Prime Plus ID System

Objective Population genetic analysis based on genetic markers harbors valuable forensic applications.In this regard,it is informative and imperative to explore Han groups as they are the largest population of China.In particular,there is a largely underrepresented amount of information from recent decades regarding the southeast costal Han Chinese.Therefore,the aim of this study is to investigate the available genetic characteristics of the Han population living in the Jinjiang,Fujian Province,Southeastern China.Methods We sampled 858 saliva samples and used the commercially available Microreader^(TM) Y Prime Plus ID System to identify population data of Y-short tandem repeat(STR)loci of this region.Results A total of 822 different haplotypes were observed.The overall haplotype diversity,discriminatory power and haplotype match probability were 0.9999,0.9999 and 0.0012,respectively.Conclusion Our results showed that the Jinjiang Han population was closely genetically related to Han groups of China.Overall,we identified a set of 37 Y-STRs that are highly polymorphic,and that can provide meaningful information in forensic practice and human genetic research.

Progress of DNA-based Methods for Species Identification

Species identification of biological samples is widely used in such fields as forensic science and food industry. A variety of accurate and reliable methods have been developed in recent years. The current review shows common target genes and screening criteria suitable for species identification, and described various DNA-based molecular biology methods about species identification. Additionally, it discusses the future development of species identification combined with real-time PCR and sequencing technologies.

Research Progress on the Integrated Detection Technology for Forensic Deoxyribonucleic Acid Genetic Markers and Ribonucleic Acid Molecular Markers

Deoxyribonucleic acid(DNA)genetic markers and ribonucleic acid(RNA)molecular markers have been widely used in forensic practices including individual identification,parentage testing,body fluid identification,determination of the age of stains,and molecular pathological diagnosis.Variant information of biological evidence and their interrelation could be revealed by the integrated detection of DNA/RNA markers.The integrated detection workflow aims to simplify working procedures,reduce time consuming and save valuable samples collected from crime scenes.Next-generation sequencing(NGS)may be an effective method for integrated DNA/RNA detection.In this review,DNA/RNA co-extraction strategies,simultaneous detection methods based on capillary electrophoresis were summarized.Research on NGS-based integrated detection methods of DNA and RNA markers was reviewed to provide a reference for forensic medicine researches and applications.

DNA recovery and analysis from skeletal material in modern forensic contexts

The generation of a DNA profile from skeletal remains is an important part of the identifica-tion process in both mass disaster and unidentified person cases. Since bones and teeth are often the only biological materials remaining after exposure to environmental conditions, intense heat, certain traumatic events and in cases where a significant amount of time has passed since the death of the individual, the ability to purify large quantities of informative DNA from these hard tissues would be beneficial. Since sampling the hard tissues for gen-etic analysis is a destructive process, it is important to understand those environmental and intrinsic factors that contribute to DNA preservation. This will serve as a brief introduction to these topics, since skeletal sampling strategies and molecular taphonomy have been dis-cussed in depth elsewhere. Additionally advances in skeletal DNA extraction and analysis will be discussed. Currently there is great variation in the DNA isolation methods used by laboratories to purify DNA from the hard tissues;however, a standardized set of short tan-dem repeat (STR) loci is analyzed by many US laboratories to allow for comparisons across samples and jurisdictions. Recent advances have allowed for the generation of DNA profiles from smaller quantities of template DNA and have expanded the number of loci analyzed for greater discriminatory power and predictions regarding the geographic ancestry and phenotype of the individual. Finally, utilizing databases and expanding the number of com-parison samples will be discussed in light of their role in the identification process.

Forensic investigation of 23 autosomal STRs and application in Han and Mongolia ethnic groups

A forensic validation study of the Early Access HuaxiaTM Platinum Polymerase Chain Reaction (PCR) kit was completed to document the performance capabilities and limitations.The genotyping of DNA samples was consistent across a large range of template DNA concentrations,with complete profiles obtained at 0.125 ng;however,no more than 2 mm× 1.2 mm punches of samples would be recommended for direct amplification.The size precision and accuracy test revealed the genotyping ability;while consistent results were obtained when comparing the kit with other commercially available systems.In addition,the whole PCR amplification can finish within approximately 45 min,making the system suitable for fastdetection.However,only partial profiles may be obtained with challenging samples,including DNA stored on Foam-Tipped Applicators (FTA) cards or some case samples.For the forensic application in ethnic groups,a total of 282 and 229 alleles were obtained in Han and Mongolia,respectively.Since the 23 short tandem repeats were independent from each other,the cumulative power of exclusion in duos was 0.999999157188 and the cumulative power of exclusion in trios was 0.999999999859 in the Han group while the cumulative power of exclusion in duos (CPEduo) was 0.999 998 848 26 and cumulative power of exclusion in trios (CPEtrio) was 0.999 999 999 79 in the Mongolia group.And good internal consistency was found between the two investigated groups and the Sichuan Han,Hui,Tibetan and Uygur according to available reference data.

Sequencing of 231 forensic genetic markers using the MiSeq FGxTM forensic genomics system-an evaluation of the assay and software

The MiSeq FGx^(TM) Forensic Genomics System types 231 genetic markers in one multiplex polymerase chain reaction (PCR) assay.The markers include core forensic short tandem repeats (STRs) as well as identity,ancestry and phenotype informative short nucleotide polymorphisms (SNPs).In this work,the MiSeq FGx^(TM) Forensic Genomics System was evaluated by analysing reproducibility,sensitivity,mixture identification and forensic phenotyping capabilities of the assay.Furthermore,the genotype calling of the ForenSeq^(TM) Universal Analysis Software was verified by analysing fastq.gz files from the MiSeq FGx^(TM) platform using the softwares STRinNGS and GATK.Overall,the performance of the MiSeq FGx^(TM) Forensic Genomics System was high.However,locus and allele drop-outs were relatively frequent at six loci (two STRs and four human identification SNPs) due to low read depth or skewed heterozygote balances,and the stutter ratios were larger than those observed with conventional STR genotyping methods.The risk of locus and allele drop-outs increased dramatically when the amount of DNA in the first PCR was lower than 250 pg.Two-person 50∶1 mixtures were identified as mixtures,whereas 100∶1 and 1000∶1 mixtures were not.Y-chromosomal short tandem repeats (Y-STRs) alleles were detected in the 100∶1 and 1000∶1 female/male mixtures.The ForenSeq^(TM) Universal Analysis Software provided the data analyst with useful alerts that simplified the analysis of the large number of markers.Many of the alerts were due to user-defined,locus-specific criteria.The results shown here indicated that the default settings should be altered for some loci.Also,recommended changes to the assay and software are discussed.

Assessment of the forensic application of 50 Y-STR markers in a large pedigree

Short tandem repeats on the Y chromosome(Y-STRs),characterized by paternal inheritance,are valuable in forensic practice.Notably,the potential application of Y-STRs in pedigrees should be drawn upon,especially in China’s surname-concentrated natural villages.The study focused on 50 Y-STRs,including 13 rapidly mutating(RM)Y-STRs that largely constitute the current Y-STR commercial kits,and determined the differences in these Y-STRs between branches in a large pedigree and the discriminatory power of these haplotypes in different units for male relatives.As indicated in the results,14 inconsistencies were observed at 9 Y-STRs between 10 father-son pairs.In addition,these 50 Y-STR haplotypes discriminated 10 out of 47 father-son pairs,106 of 148 cousin pairs,70 of 119 uncle-nephew pairs,17 of 39 brother pairs,and 14 out of 33 grandfather-grandson pairs in a large pedigree.The RM Y-STR set is able to differentiate close male relatives in a large pedigree.

Developmental validation of the novel six-dye Goldeneye^(TM)DNA ID System 35InDel kit for forensic application

Insertion/deletion polymorphisms(InDels)have been treated as a prospective and helpful genetic marker in the fields of forensic human identification,anthropology and population genetics for the past few years.In this study,we developed a six-dye multiplex typing system consisting of 34 autosomal InDels and Amelogenin for forensic application.The contained InDels were specifically selected for Chinese population with the MAF≥0.25 in East Asia,which do not overlap with the markers of Investigator^(■)DIPplex kit.The typing system was named as GoldeneyeTM DNA ID System 35InDel Kit,and a series of developmental validation studies including repeatability/reproducibility,concordance,accuracy,sensitivity,stability,species specificity and population genetics were conducted on this kit.We confirmed that the 35InDel kit is precise,sensitive,species specific and robust for forensic practice.Moreover,the 35InDel kit is capable of typing DNA extracted from forensic routine case-type samples as well as degraded samples and mixture samples.All markers are proved to be highly polymorphic with an average observed heterozygosity(He)of 0.4582.The combined power of discrimination(CPD)is 0.999999999999978 and the combined power of exclusion in duos(CPE_(D))and trios(CPE_(T))are 0.978837 and 0.999573,respectively,which are higher than those of the Investigator^(■)DIPplex kit.Thus,the GoldeneyeTM DNA ID System 35InDel kit is suitable for forensic human identification and could serve as a supplementary typing system for paternity testing.

Sequencing of human identification markers in an Uyghur population using the MiSeq FGx^(TM) Forensic Genomics System

Massively parallel sequencing(MPS)offers a useful alternative to capillary electrophoresis(CE)based analysis of human identification markers in forensic genetics.By sequencing short tandem repeats(STRs)instead of determining the fragment lengths by CE,the sequence variation within the repeat region and the flanking regions may be identified.In this study,we typed 264 Uyghur individuals using the MiSeq FGx^(^(TM)) Forensic Genomics System and Primer Mix A of the ForenSeq^(^(TM)) DNA Signature Prep Kit that amplifies 27 autosomal STRs,25 Y-STRs,seven X-STRs,and 94 HID-SNPs.STRinNGS v.1.0 and GATK 3.6 were used to analyse the STR regions and HID-SNPs,respectively.Increased allelic diversity was observed for 33 STRs with the PCR-MPS assay.The largest increases were found in DYS389II and D12S391,where the numbers of sequenced alleles were 3–4 times larger than those of alleles determined by repeat length alone.A relatively large number of flanking region variants(28 SNPs and three InDels)were observed in the Uyghur population.Seventeen of the flanking region SNPs were rare,and 12 of these SNPs had no accession number in dbSNP.The combined mean match probability and typical paternity index based on 26 sequenced autosomal STRs were 3.85E36 and 1.49Eþ16,respectively.This was 10000 times lower and 1000 times higher,respectively,than the same parameters calculated from STR repeat lengths.

Validation of the Investigator 24plex QS Kit:a 6-dye multiplex PCR assay for forensic application in the Chinese Han population

The Investigator 24plex QS Kit(QIAGEN,Hilden,Germany)is a 6-dye fluorescent chemistry short tandem repeat(STR)polymerase chain reaction(PCR)amplification system that simultaneously amplifies 20 of the expanded Combined DNA Index System(CODIS)core STR loci,SE33,DYS391,and the standard sex-determining locus,amelogenin,as well as two special internal performance quality sensor controls(QS1 and QS2),which are included in the primer mix to check the PCR performance.This study was designed to be a pilot evaluation of this STR-PCR kit in a Chinese Han population regarding the PCR conditions,sensitivity,precision,accuracy,repeatability,reproducibility,and concordance;tolerance to PCR inhibitors;applicability to real“forensic-type”samples;species specificity;mixture,balance and stutter analyses,and utility in a population investigation.The exhaustive validation studies demonstrated that the Investigator 24plex QS system is accurate,sensitive and robust for STR genotyping.In addition,these genetic markers in the population data in our study indicated that they can also be useful for forensic identification and paternity testing in the Chinese Han population.

Thanatomicrobiome composition profiling as a tool for forensic investigation

Thanatomicrobiome,or the postmortem microbiome,has been recognized as a useful microbial marker of the time and location of host death.In this mini-review,we compare the experimental methods commonly applied to thanatomicrobiome studies to the state-of-the-art methodologies in the microbiome field.Then,we review present findings in thanatomicrobiome studies,focusing on the diversity of the thanatomicrobiome composition and prediction models that have been proposed.Finally,we discuss potential improvements and future directions of the field.

Identification of a decedent in a 103-year-old homicide case using forensic anthropology and genetic genealogy

Anthropologists are often the custodians of long-term unidentified human remains though their positions as curators of university or museum skeletal collections.Various factors decrease the solvability of these legacy cases including the passage of time,the loss of provenience for specific cases,and lack of documentation or case records.While anthropologists can contribute important information toward identification,it is often necessary to explore novel and cross-disciplinary strategies to resolve difficult cold cases.In long cold cases,the postmortem interval,in particular,may be difficult to estimate leading to further challenges in achieving identification.Modern advances in radiocarbon bomb pulse dating,isotope analysis,and actualistic studies have contributed to positive identification of unidentified human remains in some legacy cases,but may not be available to all forensic practitioners and law enforcement from resource-poor agencies.Pooling resources,as well as collaborating with professionals outside of forensic anthropology,is a useful strategy to pursue when anthropological methods are exhausted.The case study presented here demonstrates a collaborative approach between forensic anthropologists,forensic genetic genealogists,and law enforcement in a century-old homicide.The dismembered and mummified parts of a male body were recovered in a remote cave in 1979 and again in 1991.Despite forensic anthropologists creating and updating the biological profile over the decades from recovery to present,no identification was made until the application of forensic genetic genealogy(FGG)to the case in 2019.New interpretations of bone microstructure and trauma analysis are presented for the case,alongside the historical documentation and“proof of life”evidence used by the genealogy team.A review of the FGG methods underscores the challenges in this case(e.g.significant endogamy,multiple aliases used by the victim)and the steps taken toward resolution.Ultimately,a combined anthropology and genealogy approach resulted in a confirmed identity for a man who was murdered in 1916.

PowerPlex■ fusion 6C system: internal validation study

This work is aimed at describing the proceedings and parameters used to validate PowerPlex■ Fusion 6C System,the polymerase chain reaction (PCR) amplification kit by Promega,for posterior implementation in the laboratorial routine of the Forensic Genetic Service.The PowerPlex■ Fusion 6C System allows multiplex PCR,through simultaneous amplification and posterior detection by fluorescence of 27 loci.Characterization of the kit was made according to the laboratory's internal validation procedure based on validation guidelines from Scientific Working Group on DNA Analysis Methods.Some parameters were evaluated,such as specificity,analytical thresholds,sensitivity,precision,mixture studies,DNA control samples,a proficiency test and changes in the PCR-based procedures: final reaction volume and cycle number,changes in the reaction mixture for direct amplification.This kit proved to be very robust and the results are in concordance with previous developmental validation by the manufacturer.In some parameters,the results were better than expected.

Epigenomic mediation after adverse childhood experiences:a systematic review and meta-analysis

Epigenetic mechanisms are potential mediators of the physiological response to abuse by altering the genetic predisposition of the cellular response to the environment,leading to changes in the regulation of multiple organ systems.This study was established to review the epigenetic mechanisms associated with childhood abuse as well as the long-term deter-minants that these epigenetic changes may have on future illness.We retrospectively ana-lysed the effect of exposure to adverse childhood experiences(ACEs,specifically those relating to childhood maltreatment)between the ages of 0 and 16years on the human epi-genome,as well as possible clinical associations.After meeting inclusion and exclusion crite-ria,36 articles were included in this systematic review.Eight of these studies did not find a relationship between childhood maltreatment and DNA methylation.Of the remaining 28 studies,nine were genome-wide association studies,whereas the rest were candidate gene studies,mainly studying effects on neuroendocrine,serotoninergic and immunoregulatory systems.Meta-analysis of correlation coefficients from candidate gene studies estimated an association of childhood adversity and DNA methylation variation at r=0.291(P<0.0001),and meta-analysis of two epigenome-wide association studies(EWASs)identified 44 differen-tially methylated CpG sites.In conclusion,childhood maltreatment may mediate epigenetic mechanisms through DNA methylation,thereby affecting physiological responses and con-ferring a predisposition to an increased risk for psychopathology and forensic repercussions.Similar evidence for somatic illnesses is not yet available.

DNA analysis of the last Brazilian unknown soldier's remains buried in Pistoia(Italy)

During World War Ⅱ,many nations took part in the war.Among the supporters of the Alliance there was also Brazil.In August 1944,under the leadership of President Getúlio Vargas,Brazil declared war on Nazi Germany and took part in the Italian campaign by send-ing many troops to support the Allies in the Central Italy.Once the conflict was over,the deceased Brazilian soldiers were buried in Pistoia,a few kilometers from Florence.But only in 1960 the Brazilian government authorized the transfer of the dead soldiers to their home-land.Five years later,during the building of the Brazilian Military Votive Monument,still in the Pistoia cemetery,a last body was found but could not be identified:so he was buried as an"unknown soldier".In December 2012,the Brazilian Embassy in Italy asked for performing forensic genetics analysis for identification purposes on the remains of this last unknown Brazilian soldier.After almost 70years a complete short tandem repeats(STR)profile was obtained,useful for any relatives searching.

Improved resolution of mixed STR profiles using a fully automated differential cell lysis/DNA extraction method

Sexual assault evidence often contains sperm cells,which are typically separated from nonsperm cells using manual differential lysis procedures.The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment.DNA yields were determined by qPCR,followed by multiplex STR amplification,CE analysis,and mixture interpretation.The automated method was capable of effective cell separation,producing DNA yields sufficient for STR amplification.Further,sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures,with 6–8fewer female alleles detected(median 1.5 alleles).Ultimately,using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains.DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium.Because of the extensive mineralisation,the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction(PCR)inhibitors.Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform.To improve the efficiency of DNA extraction from skeletal remains,the present study focuses on a modification to these already available protocols.In this study,different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol,a supplementary protocol,and a modified protocol.The modified approach included a decalcification step,whereas the Qiagen protocols worked directly on non-decalcified powder.In all three procedures,150 mg samples were used for DNA extraction.We evaluated the quantity of DNA recovered from samples,the presence of any PCR inhibitors co-extracted,the level of DNA degradation,the quality of short tandem repeat(STR)profiles,and the reproducibility of the modified procedure.When compared with the other protocols,the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors.Additionally,the STR profiles were reliable and of high quality.In our opinion,the decalcification step increases DNA recovery by softening tissues,which allows lysis solutions to act more effectively.Furthermore,the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols.These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples,such as bones and teeth.

DNA identification of compromised samples with massive parallel sequencing

Genetic profiling is a standard procedure for human identification,i.e.in criminal cases and mass disasters,and has been proven to be an important part in the process in the repatriation of victims to their relatives.In the event of a catastrophe whether it be a natural disaster,terror attack or accident,fatalities of many nationalities may be a consequence and international collaboration becomes necessary.Current DNA techniques used on a routine basis at forensic laboratories world-wide are very useful,and results reported from different labs are compared,making it possible to be matched in order to declare the identification of a victim.Statistical calculations of possibilities of a random match are achievable since population data from many parts of the world are available.However,decomposition and degradation of the remains are not uncommon in the aftermath of a catastrophe and hence it may be difficult to retrieve detailed DNA profiles from such samples.Massive parallel sequencing(MPS)is a technique capable of producing a vast amount of DNA sequence data in a high-through put manner,and panels of single nucleotide polymorphism(SNP)markers allow the amplification of small DNA fragments,often seen in compromised samples.Here,we report the results from a set of 10 samples from missing person identification cases,analyzed with an MPS based method comprising 131 SNP markers and compared with direct reference material or buccal swab samples collected from relatives of the deceased.We assess the weight of evidence of a match by statistical calculation.Furthermore,we compare results reported on different platforms using different SNP panels,and conclude that more work has to be done if results from missing person identification cases analyzed on MPS with SNP panels at different laboratories are to be fully reliable and thus comparable.

Polymorphism study of nine SNPs associated with subjective response to alcohol in Chinese Han, Hui, Tibetan, Mongolian and Uygur populations

Heavy alcohol drinking is a major public health problem,causing a large disease,social and economic burden in societies.Subjective response (SR) to alcohol is an intermediate characteristic of heavy drinking.A variety of candidate genes have been reported to be associated with SR to alcohol.In this study,we investigated nine single nucleotide polymorphisms (SNPs) related to SR to alcohol in healthy individuals from five Chinese ethnic groups,the Han,Hui,Tibetan,Mongolian and Uygur populations,and a total of 584 bloodstain samples were collected.The nine SNPs included four SNPs in alcohol-metabolizing genes (ADH1B,ADH1C,ALDH2 and CYP2E1*5B) and five SNPs in genes of neurobiological pathways (GABRA2,OPRM1,CHRNA3,HYKK and SLC6A4).A SNaPshot analysis method was developed to type these SNPs simultaneously,and all samples were typed successfully.Statistical analyses of the allele frequencies indicated that the frequencies of all SNPs,except for ADH1C,showed varying degrees of difference in the five studied ethnic groups.Tibetans showed the highest frequencies of risk alleles for heavy drinking at most loci.The genetic polymorphic differences found in this study revealed the variation in genetic susceptibility to heavy drinking in the studied populations.