Conventional Short Tandem Repeat Research Articles

Validation of next-generation sequencing-based chimerism testing for accurate detection and monitoring of engraftment in hematopoietic stem cell transplantation

Allogenic hematopoietic stem cell transplantation (allo-HSCT) is a life-saving treatment for various hematological disorders. The success of allo-HSCT depends on the engraftment of donor cells and the elimination of recipient cells monitored through chimerism testing. We aimed to validate a next-generation sequencing (NGS)-based chimerism assay for engraftment monitoring and to emphasize the importance of including the most prevalent cell subsets in proficiency testing (PT) programs. We evaluated the analytical performance of NGS-based chimerism testing (AlloSeq-HCT and CareDx) with a panel of targeted 202 informative single-nucleotide polymorphisms (SNPs) (i.e., linearity and precision, analytical sensitivity and specificity, system accuracy, and reproducibility). We further compared the performance of our NGS panel with conventional short tandem repeat (STR) analysis in unfractionated whole blood and cell-subset-enriched CD3 and CD66. Our NGS-based chimerism monitoring assay has an impressive detection limit (0.3% host DNA) for minor alleles and analytical specificity (99.9%). Pearson’s correlation between NGS- and STR-based chimerism monitoring showed a linear relationship with a slope of 0.8 and r = 0.973. The concordance of allo-HSCT patients using unfractionated whole blood, CD3, and CD66 was 0.95, 0.96, and 0.54, respectively. Utilization of CD3+ cell subsets for mixed chimerism detection yielded an average of 7.3 ± 7-fold higher donor percentage detection compared to their corresponding unfractionated whole blood samples. The accuracy of the NGS assay achieved a concordance of 98.6% on blinded external quality control STR samples. The reproducibility series showed near 100% concordance with respect to inter-assay, inter-tech, inter-instrument, cell flow kits, and AlloSeq-HCT software versions. Our study provided robust validation of NGS-based chimerism testing for accurate detection and monitoring of engraftment in allo-HSCT patients. By incorporating the cell subsets (CD3 and CD66), the sensitivity and accuracy of engraftment monitoring are significantly improved, making them an essential component of any PT program. Furthermore, the implementation of NGS-based chimerism testing shows potential to streamline high-volume transplant services and improve clinical outcomes by enabling early relapse detection and guiding timely interventions.

Direct STR profiling from laundered bloodstains: an investigation of different factors of laundering.

Bloodstains on fabrics may be washed or cleaned to eliminate incriminating evidence. These actions reduce the chances of obtaining an interpretable DNA profile. Previous studies have shown that conventional short-tandem repeat (STR) typing is affected by various factors associated with washing or laundering of stains. Here, we aim to increase the chances of obtaining interpretable STR profiles from laundered bloodstains using direct PCR. Preliminary investigations showed direct STR typing resulted in more alleles compared to conventional STR typing. We then further investigated the following factors with direct STR typing: fabric type (cotton, polyester, and denim), washing method (hand-washing and machine-washing), type of detergents (powder and liquid), washing temperature (cold to 90°C), pretreatment agents (sodium hypochlorite and hydrogen peroxide), and the number of washes (one, three, and five). Direct PCR could be successfully used for STR typing from laundered bloodstains with very high success rates. Among the three fabric types, only denim negatively affected direct STR typing, while laundering of bloodstains on cotton and polyester had a negligible effect as mostly full profiles were obtained. Multiple washes resulted in a decrease in both the numbers of alleles and peak heights. Surprisingly, washing method, type of detergent, washing temperature, and pretreatment agents only had minimal to no effect on STR profile quality. Due to the robustness and sensitivity of direct STR typing from laundered bloodstains, the method could be beneficial for violent crime investigations in forensic DNA laboratories worldwide.

Noninvasive Prenatal Paternity Testing with a Combination of Well-Established SNP and STR Markers Using Massively Parallel Sequencing.

Cell-free fetal DNA (cffDNA) from maternal plasma has made it possible to develop noninvasive prenatal paternity testing (NIPPT). However, most studies have focused on customized single nucleotide polymorphism (SNP) typing systems and few have used conventional short tandem repeat (STR) markers. Based on massively parallel sequencing (MPS), this study used a widely-accepted forensic multiplex assay system to evaluate the effect of noninvasive prenatal paternity testing with a combination of well-established SNP and STR markers. Using a ForenSeq DNA Signature Prep Kit, NIPPT was performed in 17 real parentage cases with monovular unborn fetuses at 7 to 24 gestational weeks. Different analytical strategies for the identification of paternally inherited allele (PIA) were developed to deal with SNPs and STRs. Combined paternity index (CPI) for 17 real trios as well as 272 unrelated trios was calculated. With the combination of SNPs and A-STRs, 82.35% (14/17), 88.24% (15/17), 94.12% (16/17), and 94.12% (16/17) of real trios could be accurately determined when the likelihood ratio (LR) threshold for paternity inclusion was set to 10,000, 1000, 100, and 10, respectively. This reveals that simultaneous surveys of SNP and STR markers included in the ForenSeq DNA Signature Prep Kit offer a promising method for NIPPT using MPS technology.

Evaluation of the InnoTyper21® system for the applications into trace and degraded DNA in the Korean population.

The InnoTyper 21® Human Identification kit consists of amelogenin and 20 bi-allelic Alus, retrotransposon markers existing abundantly in human genome. The InnoTyper 21® kit produces shorter amplicons (60-125 bp) than conventional short tandem repeat (STR) genotyping kit, then it is effective on the analysis of challengeable forensic samples including insufficient or highly degraded DNAs. Also, as the genotyping with InnoTyper21® kit is compatible with PCR and capillary electrophoresis, it is easy to incorporate into the workflow in forensic laboratories. In the internal validation of InnoTyper21® kit on sensitivity, degradation, and mixture studies for the evaluation in this study, we acquired full profiles on analyzing small concentration DNA (as low as 25 pg) and highly degraded DNA (up to 105 degradation index value). Through the Korean population study, forensic statistical parameters were investigated and a specific variant of T insertion in NBC51 was confirmed in six samples. Comparison of Korean population with five populations or 1000 Genomes Project data show Korean specific substructure. It is expected that the InnoTyper 21® kit will be used into the actual forensic cases, utilizing the population study investigated through this research.

Authentication, characterization and contamination detection of cell lines, xenografts and organoids by barcode deep NGS sequencing.

Misidentification and contamination of biobank samples (e.g. cell lines) have plagued biomedical research. Short tandem repeat (STR) and single-nucleotide polymorphism assays are widely used to authenticate biosamples and detect contamination, but with insufficient sensitivity at 5–10% and 3–5%, respectively. Here, we describe a deep NGS-based method with significantly higher sensitivity (≤1%). It can be used to authenticate human and mouse cell lines, xenografts and organoids. It can also reliably identify and quantify contamination of human cell line samples, contaminated with only small amount of other cell samples; detect and quantify species-specific components in human–mouse mixed samples (e.g. xenografts) with 0.1% sensitivity; detect mycoplasma contamination; and infer population structure and gender of human samples. By adopting DNA barcoding technology, we are able to profile 100–200 samples in a single run at per-sample cost comparable to conventional STR assays, providing a truly high-throughput and low-cost assay for building and maintaining high-quality biobanks.

High-Throughput Molecular Cancer Cell Line Characterization Using Digital Multiplex Ligation-Dependent Probe Amplification for Improved Standardization of in Vitro Research

Tumor cell lines are widely used for cancer research, but challenges regarding quality control of cell line identity, cross contamination, and tumor somatic molecular stability remain, demanding novel approaches beyond conventional short tandem repeat profiling. A total of 21 commonly used multiple myeloma cell lines obtained from public repositories were analyzed by digital multiplex ligation-dependent probe amplification (digitalMLPA) to characterize germline single-nucleotide polymorphisms, insertions/deletions, and somatic copy number aberrations (CNAs). Using generated profiles and an in-house developed analytical pipeline, blinded experiments were performed to determine capability of digitalMLPA to predict cell line identity and potential spike-in DNA contamination in 41 anonymized cell line samples. The dominant cell line was correctly identified in all cases, and cross contamination was correctly detected in 33 of 37 samples with spike-in DNA; there were no false-positive predictions. The four samples in which spike in was not detected all carried low levels of contamination (1%), whereas levels of contamination ≥5% were correctly identified in all cases. Unsupervised clustering of CNA profiles identified shared commonalities that correlated with initiating Ig heavy locus translocation events. Longitudinal CNA assessment of nine cell lines revealed changes under standard culturing conditions not detected by insertion/deletion profiling alone. Results suggest that digitalMLPA can be utilized as a high-throughput tool for advanced quality assurance for invitro cancer research.

Simultaneous Monitoring of Mutation and Chimerism Using Next-Generation Sequencing in Myelodysplastic Syndrome.

Monitoring minimal residual disease (MRD) provides important information during treatment of hematologic malignancies. Chimerism analysis also provides key information after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recent advances in next-generation sequencing (NGS) have enabled identification of various mutations and quantification of mutant allele burden. In this study, we developed a new analytic algorithm to monitor chimerism applicable to NGS multi-gene panel in use to identify mutations of myelodysplastic syndrome (MDS). We enrolled patients who were diagnosed with MDS and received allo-HSCT and their corresponding donors. Monitoring MRD by NGS assay was performed using 53 DNA samples by calculating mutant allele burden after treatment. For monitoring chimerism by NGS, we selected 121 single nucleotide polymorphisms (SNPs) after careful stepwise evaluation and calculated average donor allele burden. Data obtained from NGS were compared with bone marrow findings, chromosome analysis and short tandem repeat (STR)-based chimerism. SNP-based NGS chimerism analysis was accurate and even superior to conventional STR method by overcoming the various technical limitations of STR. In addition, simultaneous monitoring of mutation and chimerism using NGS could implement comprehensive pre- and post-HSCT monitoring of various clinical conditions such as complete donor chimerism, persistent mixed chimerism, early relapse, and even donor cell-derived diseases.

Single-Molecule Sequencing: A New Approach for Preimplantation Testing and Noninvasive Prenatal Diagnosis Confirmation of Fetal Genotype

We investigated the potential of next-generation sequencing (NGS) as an alternative method for preimplantation genetic testing of monogenic disease (PGT-M) with human leukocyte antigen (HLA) matching and for noninvasive prenatal diagnosis follow-up. The case involved parents who were carriers of the Fanconi anemia complementation group G (FANCG) 260delG mutation. After clinical PGT using conventional short tandem repeat and mutation analysis, two euploid disease-free embryos were transferred, resulting in a twin pregnancy. Using the original embryo whole genome amplification products from 10 embryos, NGS confirmed the genotypes of the eight nontransferred embryos for both mutation status and HLA combination. NGS also confirmed that the two transferred embryos, which resulted in a twin pregnancy, were euploid, Fanconi disease free, and HLA matched to their sick sibling. At 15 weeks' gestation, noninvasive prenatal diagnosis of the maternal cell-free DNA determined fetal fractions of 14% and 6.6% for twins 1 and 2, respectively. The maternal plasma FANCG 260delG mutation ratio was measured at 46.2%, consistent with the presence of a carrier fetus and a normal fetus. These findings provide proof of concept that NGS has clinical utility as a safe and effective PGT-M method for embryo genotyping as well as more complex direct HLA matching. In addition, NGS can be used to confirm the original PGT-M and HLA matching embryo results in early pregnancy without the need for invasive prenatal diagnosis.

Abstract LB-052: Detection of rare, deleterious clonal mutations during unrelated allogeneic hematopoietic stem cell transplants using an error correction pipeline for NGS

Abstract While next-generation sequencing (NGS) has transformed our ability to characterize the genome, it is limited in detection of rare mutations due to a high error rate of ~0.5-2.0%. We have developed an error correction pipeline and previously demonstrated that our process is capable of detecting variants as rare as 1:10,000 or 0.0001 VAF. Using our error correction pipeline, we have recently identified leukemia-related hematopoietic clones in a cohort of young healthy bone marrow donors (median age 26). In the context of allogeneic hematopoietic stem cell transplantation (HSCT), we sought to determine whether these detected leukemia-related clones of donor origin selectively engraft and correlate with transplant-related morbidities in recipients post-HSCT. Genomic profiles of pre-transplant donor samples were compared to the genomic profiles of corresponding recipient samples post-HSCT. Results show that two-thirds of the healthy donor samples harbor deleterious hematopoietic clones and that these specific clones are significantly more likely to engraft compared to benign clones. We also found that 75% of recipients who had at least one persistently engrafted, deleterious mutation developed chronic GvHD versus 50% of those without persistently engrafted clones with deleterious mutations. In comparing the results from our error correction pipeline and conventional short tandem repeat (STR) testing for donor-patient chimerism, we showed that our method provides a highly sensitive approach to both screening for donors without deleterious clones and monitoring residual disease in recipients following hematopoietic stem cell transplants (HSCT). Furthermore, the clinical implications of these results warrant a large-scale study using our error correction pipeline to detect rare variants in HSCT. Citation Format: Wing Hing Wong, Andrew Young, Todd Druley. Detection of rare, deleterious clonal mutations during unrelated allogeneic hematopoietic stem cell transplants using an error correction pipeline for NGS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-052.

Increased recovery of touch DNA evidence using FTA paper compared to conventional collection methods

Tape lifting and FTA paper scraping methods were directly compared to traditional double swabbing for collecting touch DNA from car steering wheels (n = 70 cars). Touch DNA was collected from the left or right side of each steering wheel (randomized) using two sterile cotton swabs, while the other side was sampled using water-soluble tape or FTA paper cards. DNA was extracted and quantified in duplicate using qPCR. Quantifiable amounts of DNA were detected for 100% of the samples (n = 140) collected independent of the method. However, the DNA collection yield was dependent on the collection method. A statistically significant difference in DNA yield was observed between FTA scraping and double swabbing methods (p = 0.0051), with FTA paper collecting a two-fold higher amount. Statistical analysis showed no significant difference in DNA yields between the double swabbing and tape lifting techniques (p = 0.21).Based on the DNA concentration required for 1 ng input, 47% of the samples collected using FTA paper would be expected to yield a short tandem repeat (STR) profile compared to 30% and 23% using double swabbing or tape, respectively. Further, 55% and 77% of the samples collected using double swabbing or tape, respectively, did not yield a high enough DNA concentration for the 0.5 ng of DNA input recommended for conventional STR kits and would be expected to result in a partial or no profile compared to 35% of the samples collected using FTA paper. STR analysis was conducted for a subset of the higher concentrated samples to confirm that the DNA collected from the steering wheel was from the driver. 32 samples were selected with DNA amounts of at least 1 ng total DNA (100 pg/μl when concentrated if required). A mixed STR profile was observed for 26 samples (88%) and the last driver was the major DNA contributor for 29 samples (94%). For one sample, the last driver was the minor DNA contributor. A full STR profile of the last driver was observed for 21 samples (69%) and a partial profile was observed for nine samples (25%); STR analysis failed for two samples collected using tape (6%).In conclusion, we show that the FTA paper scraping method has the potential to collect higher DNA yields from touch DNA evidence deposited on non-porous surfaces often encountered in criminal cases compared to conventional methods.

Chimerism Monitoring after Allogeneic Hematopoietic Stem Cell Transplantation Using Quantitative Real-Time PCR of Biallelic Insertion/Deletion Polymorphisms

An accurate and sensitive determination of chimerism status is mandatory after allogeneic hematopoietic stem cell transplantation. We evaluated the performance of the AlleleSEQR Chimerism Assay, which is based on quantitative real-time PCR of biallelic indel markers, using 79 recipient-donor pairs. When the informativeness of the screening panel composed of 34 markers was determined using 130 unrelated individuals, it presented a >99.9% probability of selecting at least one informative marker. The analytic sensitivity of the indel marker assay was estimated using a serially diluted DNA mixture. The detection limit of the quantitative assay approached 0.024% when 250 ng of DNA was used. We used 175 samples that had undergone hematopoietic stem cell transplantation to compare the quantitative real-time PCR assay with short tandem repeat quantitation. Results from the AlleleSEQR Chimerism Assay showed excellent correlation with those from the short tandem repeat method (r(2) = 0.931). Moreover, in some patients with relapsed leukemia, the AlleleSEQR Chimerism Assay was able to detect an early increase in recipient DNA levels that was undetectable using the short tandem repeat method. The indel marker-based AlleleSEQR Chimerism Assay can be a useful tool for following up patients after hematopoietic stem cell transplantation because of its high sensitivity compared with the conventional short tandem repeat method.

Monitoring of Hematopoietic Chimerism after Transplantation for Pediatric Myelodysplastic Syndrome: Real-Time or Conventional Short Tandem Repeat PCR in Peripheral Blood or Bone Marrow?

Quantitative real-time PCR (qPCR) has been proposed as a highly sensitive method for monitoring hematopoietic chimerism and may serve as a surrogate marker for the detection of minimal residual disease minimal residual disease in myelodysplastic syndrome (MDS), until specific methods of detection become available. Because a systematic comparison of the clinical utility of qPCR with the gold standard short tandem repeat (STR)-PCR has not been reported, we retrospectively measured chimerism by qPCR in 54 children transplanted for MDS in a previous study. Results obtained by STR-PCR in the initial study served as comparison. Because the detection limit of qPCR was sufficiently low to detect an autologous background, we defined the sample as mixed chimera if the proportion of recipient-derived cells exceeded .5%. The true positive rates were 100% versus 80% (qPCR versus STR-PCR, not significant), and mixed chimerism in most cases was detected earlier by qPCR than by STR-PCR (median, 31 days) when chimerism was quantified concurrently in peripheral blood and bone marrow. Both methods revealed a substantial rate of false positives (22.7% versus 13.6%, not significant), indicating the importance of serial testing of chimerism to monitor its progression. Finally, we propose criteria for monitoring chimerism in pediatric MDS with regard to the subtypes, specimens, PCR method, and timing of sampling.

Peripheral Blood CD34+ Specific Donor Chimerism Analysis As an Early Indicator of Disease Relapse After Allogeneic Hematopoietic Stem Cell Transplant in Patients with Myelodysplastic Syndromes and Acute Myelogenous Leukemia.

AbstractAbstract 3074 Background:Disease relapse after allogeneic hematopoietic stem cell transplant (HCT) remains a major obstacle to treatment success and cure for patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). Early identification of patients at a high risk for disease relapse has significant practical implications allowing early therapeutic interventions. We evaluated a novel approach for an early identification of a post-transplant relapse in patients with MDS and AML. Methods:CD34+ specific donor chimerism analysis was obtained in 6 HCT recipients with CD34+ AML and MDS. Starting at day +30 post HCT CD34+ cells from peripheral blood were positively selected by immunomagnetic isolation and then underwent fluorescence-activated cell sorting. Purified CD34+ cells were subsequently evaluated for percentage of donor DNA contribution by short tandem repeat (STR) analysis. CD34+ specific donor chimerism analysis was repeated monthly until hematologic relapse or death occurred. Simultaneously, conventional donor chimerism analysis was measured in the subpopulations of peripheral blood cells. Results:CD34+ cells were isolated from peripheral blood with a very high purity >95% and in sufficient quantity for reliable STR analysis in all study patients. Out of 6 evaluated patients, 3 developed hematologic relapse confirmed by bone marrow evaluation. In each relapsed patient CD34+ specific chimerism dropped to <15% prior to the confirmation of hematologic relapse. However, conventional STR analysis in granulocytes, T-cells and B-cells remained >80% (Figure 1). Moreover, obvious and steady decline in CD34+ specific donor chimerism significantly preceded the time of hematologic relapse. In contrast, no decline in CD34+ chimerism was identified in 3 patients without hematologic relapse. (Figure 1). Conclusion:Though this is a preliminary study, the results suggest that CD34+ specific donor chimerism analysis is a useful technique for timely identification of hematologic relapse in MDS and AML patients after HCT and may provide significantly earlier relapse detection than conventional chimerism analysis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Individual Identification of Racehorses from Urine Samples Using a 26‐Plex Single‐Nucleotide Polymorphism Assay

To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26-plex single-nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot(™) technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26-plex assay showed a probability of identity of 5.80 × 10(-11). Compared to the conventional short tandem repeat assay, the SNP assay used less DNA, and the rate of successful genotyping was improved to 97% using aliquots of horse urine as small as 140 μL. The urinary DNA could be successfully genotyped under proper storage concerning refrigeration or freeze-thawing. This SNP assay can be used for individual identification when suspicious results are obtained from horse doping tests.

Autosomal Short Tandem Repeat Analysis of Ancient DNA by Coupled Use of Mini‐ and Conventional STR Kits*

Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpFℓSTR(®) MiniFiler™ kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpFℓSTR(®) MiniFiler™ and Identifiler™ kits. Whereas only 21.87% of larger-sized loci profiles could be obtained with the Identifiler™ kit, 75% of the same loci profiles were determined by MiniFiler™ kit analysis. This very successful amplification of large-sized STR markers from highly degraded aDNA suggests that the MiniFiler™ kit could be a useful complement to conventional STR kit analysis of ancient samples.

High-Throughput Sequencing of Core STR Loci for Forensic Genetic Investigations using the Roche Genome Sequencer FLX Platform

The analysis and profiling of short tandem repeat (STR) loci is routinely used in forensic genetics. Current methods to investigate STR loci, including PCR-based standard fragment analyses and capillary electrophoresis, only provide amplicon lengths that are used to estimate the number of STR repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method in combination with a bioinformatic tool designed specifically to analyze sequence lengths and frequencies, we found that GS FLX STR sequence data are comparable to conventional capillary electrophoresis-based STR typing. Furthermore, we found DNA base substitutions and repeat sequence variations that would not have been identified using conventional STR typing.

Quantitative Fluorescence-Polymerase Chain Reaction Assay for the Detection of the Duplication of the Charcot Marie Tooth Disease Type 1A Critical Region

Charcot Marie Tooth (CMT) syndrome is the most common hereditary peripheral neuropathy, with an incidence of about 1 in 2500. The subtype 1A (CMT1A) is caused by a tandem duplication of a 1.5-Mb region encompassing the PMP22 gene. Conventional short tandem repeat (STR) analysis can reveal this imbalance if a triallelic pattern, defining with certainty the presence of duplication, is present. In case of duplication with a biallelic pattern, it can only indicate a semiquantitative dosage of the fluorescence intensity ratio of the two fragments. In this study we developed a quantitative fluorescence-PCR using seven highly informative STRs within the CMT1A critical region that successfully disclosed or excluded the presence of the pathogenic imbalance in a cohort of 60 samples including 40 DNAs from samples with the CMT1A duplication previously characterized with two different molecular approaches, and 20 diagnostic samples from 10 members of a five-generation pedigree segregating CMT1A, 8 unrelated cases and 2 prenatal samples. The application of the quantitative fluorescence-PCR using STRs located in the critical region could be a reliable method to evaluate the presence of the PMP22 duplication for the diagnosis and classification of hereditary neuropathies in asymptomatic subjects with a family history of inherited neuropathy, in prenatal samples in cases with one affected parent, and in unrelated patients with a sporadic demyelinating neuropathy with clinical features resembling CMT (i.e., pes cavus with hammer toes) or with conduction velocities in the range of CMT1A.

Application of SNPs in forensic casework: Identification of pathological and autoptical specimens due to sample mix-up

The analysis of single nucleotide polymorphisms (SNPs) together with conventional short tandem repeat (STR) and mitochondrial DNA (mtDNA) typing provide a forensic genetic approach for the identification of pathological and autoptical specimens in cases where the average length of DNA fragments is shorter than 150 bp in highly degraded samples. We applied a forensic genetic approach to digesta accidentally left after a training autopsy. PCR products were not amplified from samples containing the STR loci or common sequences used for mtDNA typing. The application of SNPs and deletion polymorphisms provides an alternative approach for DNA typing analysis.

Identification using DNA from skin contact: Case reports

The DNA content of different types of forensic materials can vary substantially. “Trace DNA” is the minute quantity of DNA transferred through skin contact. Here, we report three cases of identification of trace DNA using conventional short tandem repeat (STR) or mitochondrial DNA (mtDNA). DNA was successfully obtained from fabrics by swabbing or by direct extraction and subjected to STR genotyping or mtDNA typing. In two cases, there was no amplification of PCR products containing the STR loci. This indicates that the areas chosen for DNA extraction contained trace DNA and DNA from more than one source. Therefore, it is important for forensic investigators performing DNA typing to know where an item has been frequently touched by victims and/or offenders as this will influence the choice of sites on the item to be used for DNA extraction.

Development of a novel miniSTR multiplex assay for typing degraded DNA samples

A multiplex PCR was developed for the analysis of the sex-determining gene Amelogenin, four conventional STR (short tandem repeat; THO1, D18S51, D21S11 and FGA) loci with a reduced amplicon size and four miniSTR loci (D1S1677, D2S441, D10S1248 and D22S1045). A concordance study in a population of 198 Belgians revealed no differences for the conventional STR loci while a sensitivity study showed a reproducible DNA profile with as low as 30 pg of input DNA.