Effectiveness of Single‐Nucleotide Polymorphisms to Investigate Cattle Rustling

Short tandem repeats (STR)s have been the eligible markers for forensic animal genetics, despite single‐nucleotide polymorphisms (SNP)s became acceptable. The technology, the type, and amount of markers could limit the investigation in degraded forensic samples. The performance of a 32‐SNP panel genotyped through OpenArrays^TM (real‐time PCR based) was evaluated to resolve cattle‐specific forensic cases. DNA from different biological sources was used, including samples from an alleged instance of cattle rustling. SNPs and STRs performance and repeatability were compared. SNP call rate was variable among sample type (average = 80.18%), while forensic samples showed the lowest value (70.94%). The repeatability obtained (98.7%) supports the used technology. SNPs had better call rates than STRs in 12 of 20 casework samples, while forensic index values were similar for both panels. In conclusion, the 32‐SNPs used are as informative as the standard bovine STR battery and hence are suitable to resolve cattle rustling investigations.     

Pros and cons in the use of SNPs in forensic kinship investigation: a comparative analysis with STRs

Recent advances in single nucleotide polymorphisms (SNPs) research have raised the possibility that these markers could replace the forensically established short tandem repeats (STRs). In this work, we compare STRs and SNPs applicability for kinship investigation in terms of expected informative content and probability of occurrence of "difficult cases" (when isolated Mendelian incompatibilities between alleged father and child are found). Since SNPs have a much lower mutation rate than STRs, these difficulties were expected to occur less frequently if SNPs were used instead of STRs. The purpose of this paper is to make some simulations allowing the estimation of how often such difficult cases are expected to occur using both types of markers and how serious can be their impact in routine work. Our results demonstrate that a battery based exclusively on SNPs matching the informative power of current STR kits would be prone, if applied to routine paternity investigation, to the occurrence of cases where the statistical evidence would be inconclusive. We infer that the introduction of a SNP based strategy, as a substitute to the now classical STR approach poses statistical problems that must be carefully evaluated.     

Performance of the SNPforID 52 SNP-plex assay in paternity testing

The performance of a multiplex assay with 52 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was tested on 124 mother–child–father trios. The typical paternity indices (PIs) were 10⁵–10⁶ for the trios and 10³–10⁴ for the child–father duos. Using the SNP profiles from the randomly selected trios and 700 previously typed individuals, a total of 83,096 comparisons between mother, child and an unrelated man were performed. On average, 9–10 mismatches per comparison were detected. Four mismatches were genetic inconsistencies and 5–6 mismatches were opposite homozygosities. In only two of the 83,096 comparisons did an unrelated man match perfectly to a mother–child duo, and in both cases the PI of the true father was much higher than the PI of the unrelated man. The trios were also typed for 15 short tandem repeats (STRs) and seven variable number of tandem repeats (VNTRs). The typical PIs based on 15 STRs or seven VNTRs were 5–50 times higher than the typical PIs based on 52 SNPs. Six mutations in tandem repeats were detected among the randomly selected trios. In contrast, there was not found any mutations in the SNP loci. The results showed that the 52 SNP-plex assay is a very useful alternative to currently used methods in relationship testing. The usefulness of SNP markers with low mutation rates in paternity and immigration casework is discussed.     

Using STR,MiniSTR and SNP markers to solve complex cases of kinship analysis

In complex kinship investigation, miniSTRs and SNPs have been frequently used in order to increase the likelihood ratio (LR), when the results obtained for the most commonly used STR multiplexes were not informative enough. In this work, we describe the results obtained when using a battery of 23 STRs, 3 miniSTRs and 52 SNPs to investigate three complex paternity cases where the father was not available, and one paternity case with bone samples, from which no results could be obtained for STRs (including the 3 miniSTRs, D10S1248, D14S1434 and D22S1045). In all cases, the additional information provided by the SNPforID 52plex identification panel was enough to achieve conclusive results.     

MtSNP typing before mtDNA sequencing: Why do it?

Analysis of control mitochondrial DNA (mtDNA) hypervariable regions is sometimes the only available method to study hair evidence in forensic casework although being a laborious technique. Nowadays there is a huge interest in new genetic markers such as single nucleotide polymorphisms (SNPs) to type degraded forensic samples. For that purpose, a 10-Plex mitochondrial SNP for haplogroup typing, chosen from several SNP studies and useful to study the most common populations in our laboratory was applied in forensic casework. Hair shafts from three forensic cases with different ethnic backgrounds were studied with mtDNA sequencing and compared with mitochondrial SNPs (mtSNPs) study. Coding mtSNP typing prior to sequencing can allow for a rapid screening in forensic casework, which is emphasized in the first two cases. Moreover, in cases in which mtDNA sequencing fails, mtSNPs can still be detected. This 10 SNP loci multiplex provides a less expensive and simpler method for mitochondrial typing compared to control region mtDNA sequencing, especially when used as a fast screening method.     

Internal validation of 29 autosomal SNP multiplex using a ABI 310 genetic analyser

In the last few years genetic identification and paternity testing have begun to make increasing use of autosomal SNP (Single Nucleotide Polymorphism) typing as a supplement or alternative to STR analysis. With the improvement in detection technology SNP analysis is likely to be easier and more sensitive, with the generation of new methods and multiplex systems for a growing array of SNP markers. SNPforID consortium developed 52 SNP PCR multiplex for human identification purposes detected with 23 plex and 29 plex single base extension reactions (Auto1 and 2 respectively). In this study, internal validation for the 29 SNPs of Auto2 was carried out by performing a 29 plex PCR and single base extension reaction on control samples and previously analyzed forensic casework and subsequent detection with an AB 310 Genetic Analyzer. We tested the accuracy, precision, sensitivity and reproducibility of the Auto2 multiplex with this instrument in our laboratory. We used 9947A control DNA samples of the AmpF?STR Identifiler™ kit to test the validation parameters together with non-probative DNA samples from whole blood and buccal swab samples of 29 healthy donors from different parts of Istanbul. Good results were obtained but interpretation of the peak patterns obtained on the AB 310 requires care and thorough optimization before they can be readily compard to those obtained from multiple capillary AB 31xx Analyzers. We succesfully optimized and validated the SNPforID Auto2 multiplex system for identification analyses in our laboratory.     

Significance of additional RFLP single locus probes analysis after STR based determination of sibship

Autosomal STR typing alone seems to be no sufficient tool for resolving deficiency cases (e.g. cases of questioned paternity or half-sibships). Therefore, we investigated whether the additional analysis of RFLP single locus probes can improve the solution of such complicated kinship cases. We analyzed 207 children and men from 101 families using the AmpFlSTRIdentifiler multiplex PCR kit and three RFLP single locus probes. A comparison between each child and all unrelated men resulted in 11,023 man / child pairs. Less than three excluding STRs were found in 125 child / unrelated man pairs (1.13%). Additional analysis of RFLP results reduced the number of ambiguous cases to 35. Half-sibling pairs were simulated using STR results from 20 cases with high paternity probabilities (group 1) and relatively low paternity probabilities (group 2). Using a commercially available computer program we calculated probabilities for 778 half-sibling pairs. In 35 pairs (4.49%) half-sibling probabilities over 90.0% could be calculated. Additional investigation of RFLP single locus probes did not lead to a more reliable evaluation of these results. The combined investigation of autosomal STRs and RFLP single locus probes can satisfactorily solve deficient paternities but does not contribute to the solution of questioned half-sibships.     

Resolving relationship tests that show ambiguous STR results using autosomal SNPs as supplementary markers

When using a standard battery of STRs for relationship testing a small proportion of analyses can give ambiguous results – where the claimed relationship cannot be confirmed by a high enough paternity index or excluded with fully incompatible genotypes. The majority of such cases arise from unknowingly testing a brother of the true father and observing only a small number of exclusions that can each be interpreted as one- or two-step mutations. Although adding extra STRs might resolve a proportion of cases, there are few properly validated extra STRs available, while the commonly added hypervariable SE33 locus is four times more mutable than average, increasing the risk of ambiguous results. We have found SNPs in large multiplexes are much more informative for both low initial probabilities or ambiguous exclusions and at the same time provide a more reliable genotyping approach for the highly degraded DNA encountered in many identification cases. Eight relationship cases are outlined where the addition of SNP data resolved analyses that had remained ambiguous even with extended STR typing. In addition we have made simulations to ascertain the frequency of failing to obtain exclusions or conclusive probabilities of paternity with different marker sets when a brother of the true father is tested. Results indicate that SNPs are statistically more efficient than STRs in resolving cases that distinguish first-degree relatives in deficient pedigrees.     

联合应用常染色体STR和X-STR标记鉴定单亲疑难案例

目的通过对常染色体和X染色体遗传标记的检测,探讨单亲疑难案例的鉴定策略。方法提取3个单亲鉴定案例的6份血样,采用Goldeneye 20A试剂盒和AGCU21＋1试剂盒检测常染色体上39个STR基因座,采用自主研制的16重X-STR扩增系统检测X染色体上16个STR基因座。结果用Goldeneye 20A试剂盒检测后发现每个单亲案例均有一个基因座不符合遗传规律,当常染色体STR基因座增加到39个时,案例1累计出现3个矛盾基因座;案例2和案例3均没有出现新的矛盾基因座。X染色体STR分型结果显示案例1有8个矛盾基因座,案例2和案例3无矛盾基因座,与常染色体分型结论相符。结论对于出现单基因座不符合遗传规律的母女、母子、父女单亲案例鉴定,不仅可以增加新的常染色体STR检测,也可以增加X染色体STR的检测,这样在相互验证的同时也能获得更加可靠的鉴定意见。     

Autosomal and Y-STR mutations in Lithuanian population

The new approach is given in this work combines autosomal and Y-chromosomal short tandem repeats (STRs) data in paternity testing with father/son pairs taking into account mutation events. Our results show that Y-STRs can identify nonpaternity cases with high accuracy but need to be validated with autosomal STR to establish paternity. Autosomal and Y-STR mutations give disputing questions in paternity testing as well.     

Forensic Identification Using a Multiplex Assay of 47 SNPs*

Abstract: As a powerful alternative to short tandem repeat (STR) profiling, we have developed a novel panel of 47 single nucleotide polymorphisms (SNPs) for DNA profiling and ABO genotyping. We selected 42 of the 47 SNPs from a panel of 86 markers that were previously validated as universal individual identification markers and identified five additional SNPs including one gender marker and four ABO loci. Match probability of the 42 validated SNPs was found to be 9.5 × 10^?18 in Han Chinese. SNP analysis correctly assessed a panel of historical cases, including both paternity identifications in trios and individual identifications. In addition, while STR profiling of degraded DNA provided information for 11 loci of 16 potential markers with low peak intensities, SNPstream^® genotyping was sufficient to identify all 47 SNPs. In summary, SNP analysis is equally effective as STR profiling, but appears more suited for individual identification than STR profiling in cases where DNA may be degraded.     

常染色体STR突变基因座父权指数计算

目的概括归纳常染色体STR突变基因座的父权指数计算方法,以在实际检案中应用推广。方法根据目前对常染色体STR基因座突变的认识,用经验递减模型从基因座突变率计算等位基因突变率,分别推导在标准三联体和二联体亲子鉴定中STR突变基因座的父权指数计算式,并举例进行演算。结果总结了在标准三联体鉴定中,只允许假设父突变和既允许假设父也允许母发生突变时,以及二联体鉴定时X和Y的计算式。结论STR基因座发生突变时计算得的父权指数明显低于未发生突变时,提示要检测更多的遗传标记才能使累积父权指数达到认定亲权关系的标准。     

SNP genotyping of forensic casework samples using the 52 SNPforID markers

The analysis of degraded DNA is one of the biggest challenges in forensic casework. SNPs, which can be amplified using small amplicons, have previously been successfully applied to the profiling of forensic evidence that could not be analyzed using conventional STRs. Here we selected the 52 SNPforID SNP markers, with amplicons that ranged in size from 59 bp to 115 bp, and used them to profile a range of casework samples from Malaysia. DNA degradation is a common problem in Malaysia due to the high temperatures and humidity. To carry out the study we modified the 52 SNPforID markers into four 13-plex SNaPshot assays to enable easier interpretation of profiles on the ABI PRISM^® 310 and 3500.Fifty-one crime samples comprising bloodstains on cloth, swabs, and a mat and 2 swabs of trace DNA from 10 crime scenes in Malaysia were profiled after DNA extraction using a phenol–chloroform method. The samples were also subjected to STR analysis using the Powerplex^® 16 system (Promega), which resulted in only 17 full profiles and 9 partial profiles; using SNPs, 36 full profiles and 5 partial profiles could be generated.     

A multipurpose panel of microhaplotypes for casework

Microhaplotypes (MHs) are SNP-based multiallelic loci that have several advantages over individual SNPs and short tandem repeats (STRs). For several years we have been searching for better MHs based on the effective number of alleles at a locus (A_e) and the locus informativeness for population relationships (I_n) with thoughts of incorporating MHs into casework. We genotyped a multiplex of our best 90 MHs on 79 populations. We have ranked the 90 by A_e and analyzed the top 24 to evaluate their potential value in forensic casework. We chose 24 to compare with the popSTR dataset of 20 CODIS markers plus four other STRs commonly typed. PopSTR has full data on 32 populations; our 24 MHs have full data on 79 pops. We have compared the two sets of 24 loci (MH and STR) in four areas: individualization, biogeographic ancestry, kinship analysis, and mixture resolution.     

基于高通量测序进行无创产前亲子鉴定的可行性

目的初步探讨基于高通量测序进行STR分型的技术方法应用于无创产前亲子鉴定的可行性。方法选择13个STR基因座(6个常染色体STR基因座,6个Y染色体STR基因座,1个性别判定基因座),进行复合PCR扩增和高通量测序文库构建后,采用Ion PGM400高通量测序平台进行测序,并采用自主研发软件NGS-STR genotyper(perl脚本)进行STR分型,本文简称上述过程为NGS-STR分型。对13个母子配对混合样本(母亲:儿子=2%~50%)、1组家系样本进行了上述NGS-STR分型,旨在(1)了解其在混合样本中的灵敏度及分型情况;(2)了解其在无创产前亲子鉴定中的应用可能性。结果 (1)当混合样本中低组分(儿子)的比例超过8%,所有基因座均可检出低组分的STR信息;(2)对1例血浆样本进行NGS-STR分型,共计69.2%的基因座可检出胎儿的STR基因型信息,且所有检出基因座均符合孟德尔遗传规律。结论初步证明了NGS-STR分型技术具有进行无创产前亲子鉴定的可行性。     

Paternity investigation experience with a 40 autosomal SNP panel

With both the SNPforID and the Kidd panels of autosomal SNPs available, we selected the 40 most informative and population-independent SNPs from both these sets for evaluation in paternity testing and as a prelude for forensic human identification.We used the published primer sequences and constructed PCR multiplexes for genotyping using the SNaPshot assay. Fifty trios and 50 duos previously analysed using conventional autosomal STR markers were re-analysed using the 40 SNPs. We report our findings regarding the practical use of these markers including unexpected mutations which impacted significantly on the use of this panel.     

SNPs and MALDI-TOF MS: tools for DNA typing in forensic paternity testing and anthropology

DNA markers used for individual identification in forensic sciences are based on repeat sequences in nuclear DNA and the mitochondrial DNA hypervariable regions 1 and 2. An alternative to these markers is the use of single nucleotide polymorphisms (SNPs). These have a particular advantage in the analysis of degraded or poor samples, which are often all that is available in forensics or anthropology. In order to study the potential of SNP analysis in these fields, 41 SNPs were selected on the basis of following criteria: conservation, lack of phenotypic expression, and frequency of occurrence in populations. Thirty-six autosomal SNPs were used for genotyping 21 inclusionary and 3 exclusionary paternity cases. The behavior of 5 X-chromosome SNPs was analyzed in a French representative population. Our approach to SNP typing is a multiplex PCR based amplification followed by simultaneous detection by primer extension (PEX) analyzed by Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The selected autosomal SNPs showed independent inheritance and gave clear results in paternity investigation. All X-SNPs were useful as both paternity and identification markers. PEX and MALDI-TOF MS, with their high sensitivity, precision and speed, gave a powerful method for forensic and anthropological exploitation of biallelic markers.     

Supplementary markers for deficient immigration cases: Additional STRs or SNPs?

Analysis with commonly available STR kits can sometimes fail to produce sufficient information in immigration cases containing only one parent. In these cases, not only does paternity/maternity need to be assured, but also other possible relationships dismissed (e.g. avuncular relationships).We have taken more than 50 of these cases and investigated which type of additional marker produces the greatest benefits: 48 SNPs or 6 additional informative STRs (including 5 additional markers from the new European extended set). The results of this analysis show the SNPs to be of greater value.     

亲子鉴定STR突变的考虑

亲子鉴定中常会发生STR（short tandem repeats,短串联重复）突变的情况。突变对亲子关系的判定会造成困扰。对STR突变规律和亲子鉴定中所遇到的STR突变问题、突变的判定、突变下非父排除率和父权指数的计算以及需与突变区分的情形等问题进行综述和讨论。     

STR and SNP analysis of human DNA from Lucilia sericata larvae's gut contents

Importance of forensic entomology becomes inevitable when come across some incident where corpse is unidentifiable and lot of maggots or other insects are present. The most common application of forensic entomology is to use insects for the identification of specimens or human remains. DNA analysis recovered from a larva's gut contents can be used to identify a missing body. The obtained human STR and SNP profile support the association of a maggot to a specific patients or corpse. Main aim of this research was the identification of human DNA from gut contents of third instar maggots (larvae of Lucilia sericata) placed on diabetic patient's wounds for treatment purpose. Maggots (8–15) were taken from each diabetic patients (no. of the patients 8) and DNA was extracted from the gut contents manually by using Qiagen tissue protocol. Agarose gel electrophoresis was performed and the total size of DNA was seen using UV transilluminator. PCR amplification, STRs and SNPs profiling was then performed using PCR 9700 and AmpFLSTR Identifiler and SnaPshot Multiplex Kit (Applied Biosystems) respectively. The results were analyzed on ABI 310. SNP profiles were good and identifiable compared to the STRs where amplification was poor and the peaks were low. This may be the fact of the enzymatic activity present in the gut of the larvae which cause tremendous reduction in DNA size and thus yield. The results of this study reveals that it is possible to obtain a complete human profile using STRs and SNPs even if DNA is recovered from gut contents of maggots.