Multiplex STR Systems Research Articles

Genetic Polymorphisms and Forensic Parameters of Thirteen X-Chromosome Markers in the Iraqi Kurdish Population.

X-chromosome short tandem repeat (X-STR) tools are crucial in forensic genetics and human population fields. This study presents the development and validation of a multiplex STR system consisting of thirteen X-STR loci and amelogenin specific to the human X chromosome. The system was optimized and tested for species specificity, sensitivity, stability, and DNA mixture using 9947A female and 9948 male control genomic DNA. The amplified products of nine loci were sequenced to determine the correct amplicon length. Allele frequencies, forensic parameters, mean exclusion chance (MEC), linkage disequilibrium (LD), and allelic patterns were investigated using DNA samples from 225 (159 male, 66 female) unrelated Kurdish individuals who live in Sulaymaniyah province in the Kurdistan region of Iraq. The most informative locus in the Kurdish population was GATA172D05, while the least informative locus was DXS10164. The results demonstrated that the 13 X-STR system is highly polymorphic and sensitive for forensic DNA identification. Genetic distance-based clustering, metric multidimensional scaling (MDS), and correlation matrix were analyzed for 19 ethnic groups and populations. The phylogenetic tree showed that populations clustered according to their ethnogeographic relationships. The findings revealed genetic links between the Iraqi Kurds, Caucasians, Iraqi Arabs, United States (U.S.) ethnic groups, and Chinese populations.

Front Cover: Evaluation of microfluidics‐based droplet PCR combined with multiplex STR system in forensic science

Front Cover: Evaluation of microfluidics‐based droplet PCR combined with multiplex STR system in forensic science

Evaluation of microfluidics-based droplet PCR combined with multiplex STR system in forensic science.

Because of its excellent monodispersity, high throughput, and low volume, microfluidics-based droplet PCR has become the core technology of digital PCR, next-generation sequencing, and other technology platforms. This study constructed a microfluidic water-in-oil droplet PCR system and amplified a commercially available forensic 22-plex short tandem repeat detection system. We analyzed the sensitivity, concordance, amplification efficiency of the droplet PCR, and influence factors of the above aspects. The droplet PCR showed high concordance with conventional bulk PCR and had high sensitivity as 0.125ng. Furthermore, we observed the performance of droplet PCR in high-order mixed DNA. As the mixture ratios from 10:1 to 30:1, droplet PCR presented more mixture proportion (Mx) increased loci from 11 (57.89%) to 17 (89.47%). In the mixture ratios 20:1, 25:1, and 30:1, significant Mx differences between droplet PCR and bulk PCR were observed (p <0.05). The results showed that the droplet PCR could improve the identification of the minor contributor's DNA in a two-person mixture and alleviate the imbalanced amplification problem. This study provides a reference and basis for the wide application of droplet PCR in forensic science.

Forensic evaluation of SE33 marker in Indian population using Global Filer, Fusion 6 c and Panglobal autosomal STR multiplex

Capillary electrophoresis based forensic DNA typing using PCR with multiplex STR kit is gold standard now. But even one mismatch in the studied markers results in an inconclusive report. This deficiency is suggested to be taken care of by using alternative markers and/or by increasing the number of STR markers. Three new generation multiplex autosomal STR systems namely GlobalFiler™ PCR Amplification Kit (Thermo Fisher Scientific Foster City, CA, USA), PowerPlex Fusion 6C (Promega, Madison, WI, USA), PanGlobal Human DNA Identification Kit (Health Gene Technologies Co., Ltd) incorporating new 20 CODIS and the SE33 marker were used on 819 samples to observe the forensic performance of SE33 marker. The study presents first forensic evaluation of SE33 marker included in new generation multiplex systems being used for forensic DNA typing. A total of 52 alleles were observed on the SE33 marker in the studied population. Forensic parameters including allele frequency, Power of discrimination, Polymorphism information content, Power of exclusion, Paternity index, Observed and Expected Heterozygosity, Matching Probability, Shannon's Information and Index and Fixation Index are reported for the studied marker.

COMPARATIVE ANALYSIS OF THE EFICENCY OF POWERPLEX 16 AND POWERPLEX FUSION MULTIPLEX STR KITS IN THE ANALYSIS OF THE CHALLENGING FORENSIC SAMPLES

Since the introduction of the term low copy number DNA, also referred as low template DNA, touch DNA or trace DNA analysis, it has quickly become focal point of forensic DNA testing as well as other DNA based studies. Low template DNA (ltDNA) samples can be described as the samples which involve single source samples with template DNA in concentrations below 100 picograms (pg). Due to sensitivity of ltDNA samples to contamination, it is of great importance to optimize performance of the multiplex STR systems and existing protocols to increase chance of successful analysis. The main objective of this study was analysis of 20 challenging samples (skeletal remains, cigarette buts, chewing gum, poorly collected buccal swabs etc.) mostly low template DNA samples, preliminarily profiled by PowerPlex® 16 multiplex STR systems and additionally processed with new generation multiplex STR kit PowerPlex® Fusion. Sample isolation was done using a standard phenol-chloroform method for bone samples and DNeasy® Blood and Tissue Kit for other forensic samples. PowerPlex® 16 (PP16), multiplex STR system and PowerPlex® Fusion (PP Fusion) were used for co-amplification of 15 and 24 autosomal STR loci respectively. Results of this preliminary study suggest that PP Fusion primer set is better optimized for the analysis of ltDNA samples, and it is more robust regarding presence of the potential PCR inhibitors.

Chimerism Analysis after Haploidentical Stem Cell Transplantation: Is It Necessary for All Patients?

Introduction: Allogenic hematopoietic stem cell transplantation (SCT) is often the only curative treatment for a part of patients with malignant or benign hematological disorders. The availability of a HLA matched related or unrelated donor remains a major obstacle which can be resolved by the presence of alternative donors, like umbilical cord blood, partially matched unrelated or haploidentical family donors. T cell repleted SCT using haploidentical donors (H-SCT) has considerably improved over the last years due to better control of Graft Versus Host Disease (GVHD) using post-transplantation cyclophosphamide (PTCY).Our study aims to shed light on the interest of chimerism evaluation after H-SCT and to elucidate the cause of graft failure (GF) in our H-SCT recipients.Patients and methods: We conducted a retrospective study of 179 patients (pts) who received a H-SCT in our center from august 2009 to march 2016. The pts characteristics are reported in Table 1 and 2. One hundred sixty pts received a Reduced Intensity (RIC) and 19 pts a MyeloAblative Conditioning (MAC) regimen. Twenty-eight pts had refractory or relapsed acute myeloid leukemia. All pts received PTCY on days 3 and 4 and Ciclosporine A and mycophenolate of mofetil since day 5 for GVHD prophylaxis. Twenty pts received H-SCT for relapse after a first SCT with a HLA identical donor. Stem cell sources were bone marrow (BM) for 19 pts, peripheral blood stem cells (PBSC) for 157 pts and BM+PBSC for 3 pts. The donors were children (79 daughters sons), parents (4 fathers, 14 mothers), family members (75 siblings, 1 cousin, 1 nephew) and 5 unknown.Chimerism analysis: The peripheral blood CD3 positive cells were selected using the kit Human CD3 Positive selection (Stemcell) and genotyping was performed with the PowerPlex 18D System (Promega), a multiplex STR system allowing the co-amplification of 18 loci.DSAs (donor-specific anti-HLA antibodies): The DSAs were identified with the use of highly sensitive solid-phase immunoassays. Desensitization therapy with Bortezomib, Rituxan and plasmapheresis was performed in two patients with a high level of DSAs.Results: Chimerism could be evaluated in 167 patients, where 162 had ≥ 98% of donor cells at a median time of 35 d post-transplant (range 15-170) without secondary GF.One patient suffering from sickle disease had stable mixed chimerism (82% donor). Only 4 pts had primary GF (2%), all of donor-recipient pairs were ABO compatible, all of them received a Baltimore conditioning whereas 2 pts had BM as SC source. Recipients with PBSC as SC source had poor CD34+ cells infused (1,2 and 2,3 x 106/kg). One of the 4 pts had a high level of DSAs. A patient with mycosis fungoide had secondary GF after relapse (PBSC/DSAs negative).One patient with a high level of DSAs (78%) had a primary graft failure. Two pts with a high level of DSAs who were desensitized by the described procedure. Forty-five patients had a positive anti-HLA antibody no specific to donor with a median level of 3% (range: 1-45%) with no impact on engraftment (Anti-HLA antibody class I in 30 pts, class II in 11 pts and mixed in 4 pts).The median number of CD34+ cell count was 5,2x 106/kg (range: 1,3-17) in 160 pts who had PBCS as SC source.With a median follow up of 532 d (range: 164-1587d), 123 pts are alive and 116 of them are in CR.Conclusion: Full donor chimerism is obtained in almost all pts after T cell repleted H-SCT with PTCY. Primary graft failure occurred principally in pts with high levels of DSAs and poor graft CD34+ cells. Chimerism analysis post H-SCT is not crucial and might focus on a small group of high-risk pts. Detection of DSAs is crucial in HLA mismatched transplants not only to select the most appropriate donor but also to include a pre-transplantation strategy of desensitization to minimize the risk of graft failure. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combined effects of multiple linked loci on pairwise sibling tests.

The advanced multiplex STR system, PowerPlex Fusion, includes four linked locus pairs. The conventional Identifiler system has one pair of linked loci. Therefore, sibling tests conducted using the advanced system might be more affected by linkage than those conducted using the conventional system. This study simulated single and combined effects of the four linked locus pairs on pairwise sibling tests. Simulated genotypes of 100,000 pairs of full siblings and nonrelatives were constructed according to allele frequencies of the Japanese population. The single linkage effect was evaluated for simulated genotype data by calculating both the likelihood ratio accounting for the linkage between two loci and the likelihood ratio ignoring the linkage. The combined effect was obtained by multiplication of the respective single effects. Furthermore, we investigated the possibility that ignoring the linkage affects subject classification by introducing a scale of the likelihood ratio into sibling tests. The single effect in the Identifiler analysis was 0.645-1.746 times if the linkage was ignored. Overestimations and underestimations were predictable from the identical-by-state status at two linked loci. The combined effect in the PowerPlex Fusion analysis was 0.217-7.390 times. Ignoring the linkage rarely caused a false conclusive or inconclusive result, even from PowerPlex Fusion analysis. Application of the advanced system improved sibling tests considerably. The additional examined loci were more beneficial than the adverse effect of the linkage derived from the four linked locus pairs.

Paternally inherited trisomy at D21S11 and mutation at DXS10135 microsatellite marker in a case of fetus paternity establishment

The case of establishment of paternity of an aborted fetus was examined with 15 autosomal STR markers. The genotype of the fetus was X at amelogenin marker and showed inheritance of both the alleles of father at D21S11 marker, thus displaying unusual tri-allelic pattern. The cases where mutation in any of biparental autosomal STR markers is observed, the use of additional STR marker system is recommended. On testing all the three samples with 12 X-STR markers, all the maternal and paternal alleles were accounted in the female fetus except at DXS10135 marker. The genotypes of mother, fetus and father at DXS10135 were 20, 22; 20, 20 and 21, which confirmed mutation of the paternal allele in the female fetus. The paternal allele contracted from 21 to 20. The allele peak heights of D21S11 and DXS10135 markers were also examined to rule out the possibility of any false allele. The probability of paternity was 0.99999999, which confirmed the paternity of the fetus. This paper presents unusual occurrence of mutation observed with two multiplex STR systems, with AmpflSTR identifier plus Kit (Applied Biosystems, Foster City, CA) and Investigator Argus X-12 multiplex kit (Qiagen, Germany), thus suggesting forensic DNA experts on high alert while interpreting the DNA test results.

High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6–200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250pg of input DNA, and 62pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.

Comparative performance between “next generation” multiplex systems and the new European Standard Set of STR markers in the Portuguese Population

Various multiplex STR systems have been developed by the major commercial companies in the forensic genetics field to comply with the recent establishment of the global European Standard Set (ESS) of markers. Of the various alternatives available, our laboratory decided to test the recent ESSplex Plus system (Qiagen) and the NGM kit (Life Technologies), which share the same 15 STR loci and comprise the most recently established ESS markers (D1S1656, D2S441, D10S1248, D12S391 and D22S1045). Apart from evaluating the kits' technical performances, a population and segregation study was carried out on a Portuguese sample, with the aim of introducing the ESS markers in routine forensic casework. A total of 370 individuals were sampled for this purpose, comprising 120 true trios (125 fathers, 125 mothers and 120 sons/daughters). No deviations from Hardy-Weinberg equilibrium were detected for the five new loci in the Portuguese population and no genotyping inconsistencies were observed between kits. Parameters of forensic interest revealed that, of the five ESS markers, D1S1656 was the most informative in our sample. Comparison of performances between all autosomal multiplex systems available in our laboratory (ESSplex Plus, NGM, Identifiler Plus and Powerplex 16 HS), revealed that the multiplex kits with the ESS markers generally showed better performances and, among these, the ESSplex Plus kit showed slightly higher sensitivity and a better detection of degraded DNA information.

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpF lSTR ® Idenitfiler ® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.

Study of the genetic relationship and diversity patterns in the Azores based on 15 STR markers

The Azores, the largest Portuguese archipelago, is composed of nine islands unevenly distributed by three groups: the Eastern group (São Miguel and Santa Maria), the Central (Terceira, Pico, Faial, São Jorge and Graciosa) and the Western group (Flores and Corvo). Here, we describe the genetic diversity patterns for each Azorean island and their genetic relationship based on a total of 554 samples. Genotyping was carried out by the multiplex STR system PowerPlex ® 16 (Promega). The results demonstrate that the average gene diversity values vary between 0.768 and 0.797 for Corvo and Terceira, respectively. The comparison of these data with mainland Portugal (0.765) reveals higher values for the Azorean islands. The dendrogram shows that the most distantly apart are Flores and Corvo; however, there is no genetic differentiation between all islands. Moreover, we observe a clustering of the island's populations by their geographical and socio-cultural proximity. On the other hand, historical records mention a differential settlement between islands. The results show that, nowadays, there are no regional genetic differences in the archipelago due to internal migration. Taken together, the knowledge here obtained will provide insights about the allelic structure of health-related genetic variation in the Azorean population.

DNA Typing from Forensic Mixed Samples by Peak Height Ratios on Multiplex STR Systems

DNA Typing from Forensic Mixed Samples by Peak Height Ratios on Multiplex STR Systems

Evaluation of “in house” criteria for PCR-based analysis in immigration casework

“In house” criteria for PCR-based analysis in immigration casework have been evaluated in 28 immigration families. The isolated DNA was amplified using the multiplex systems SGM+ (Applied Biosystems), CTTv, FFFL and PowerPlex 16 (Promega). PCR products were separated on the ABI 310 analyzer. The one and two parental indices ( I) were calculated using the DNA.VIEW software package (C. Brenner, Oakland, USA). DNA was isolated twice to reduce mistakes. Combining the results of the multiplex STR systems, used in this evaluation, appeared to be successful in analysing immigration casework. From the results of this evaluation, it appeared that the involvement of other family members in immigration casework has to be critically evaluated.

Validation of short tandem repeat analysis for the investigation of cases of disputed paternity

This study details validation of two separate multiplex STR systems for use in paternity investigations. These are the Second Generation Multiplex (SGM) developed by the UK Forensic Science Service and the PowerPlex 1 multiplex commercially available from Promega Inc. (Madison, WI, USA). These multiplexes contain 12 different STR systems (two are duplicated in the two systems). Population databases from Caucasian, Asian and Afro-Caribbean populations have been compiled for all loci. In all but two of the 36 STR/ethnic group combinations, no evidence was obtained to indicate inconsistency with Hardy-Weinberg (HW) proportions. Empirical and theoretical approaches have been taken to validate these systems for paternity testing. Samples from 121 cases of disputed paternity were analysed using established Single Locus Probe (SLP) tests currently in use, and also using the two multiplex STR systems. Results of all three test systems were compared and no non-conformities in the conclusions were observed, although four examples of apparent germ line mutations in the STR systems were identified. The data was analysed to give information on expected paternity indices and exclusion rates for these STR systems. The 12 systems combined comprise a highly discriminating test suitable for paternity testing. 99.96% of non-fathers are excluded from paternity on two or more STR systems. Where no exclusion is found, Paternity Index (PI) values of > 10,000 are expected in > 96% of cases.

Validation of short tandem repeat analysis for the investigation of cases of disputed paternity

This study details validation of two separate multiplex STR systems for use in paternity investigations. These are the Second Generation Multiplex (SGM) developed by the UK Forensic Science Service and the PowerPlex™1 multiplex commercially available from Promega Inc. (Madison, WI, USA). These multiplexes contain 12 different STR systems (two are duplicated in the two systems). Population databases from Caucasian, Asian and Afro-Caribbean populations have been compiled for all loci. In all but two of the 36 STR/ethnic group combinations, no evidence was obtained to indicate inconsistency with Hardy-Weinberg (HW) proportions. Empirical and theoretical approaches have been taken to validate these systems for paternity testing. Samples from 121 cases of disputed paternity were analysed using established Single Locus Probe (SLP) tests currently in use, and also using the two multiplex STR systems. Results of all three test systems were compared and no non-conformities in the conclusions were observed, although four examples of apparent germ line mutations in the STR systems were identified. The data was analysed to give information on expected paternity indices and exclusion rates for these STR systems. The 12 systems combined comprise a highly discriminating test suitable for paternity testing. 99.96% of non-fathers are excluded from paternity on two or more STR systems. Where no exclusion is found, Paternity Index (PI) values of >10 000 are expected in >96% of cases.

New reference allelic ladders to improve allelic designation in a multiplex STR system.

This paper reports the composition of a new reference allelic ladder mixture for use with a multiplex DNA profiling system consisting of six short tandem repeat loci. The loci included in this mixture are HUMTH01, D21S11, D18S51, D8S1179, HUMVWAF31/A, HUMFIBRA/FGA and an amelogenin sex test. Sequence analysis of individual ladder alleles was carried out and allelic designations made in accordance with the recommendations of the International Society of Forensic Haemogenetics (1992; 1994). A series of rare alleles which increase the range of alleles previously reported were identified. By including some of the rare alleles into the ladder marker system, we have significantly improved the ability to identify new alleles in unknown samples.