Evaluation of Storage Conditions and the Effect on DNA from Forensic Evidence Objects Retrieved from Lake Water.

DNA analysis of traces from commonly found objects like knives, smartphones, tapes and garbage bags related to crime in aquatic environments is challenging for forensic DNA laboratories. The amount of recovered DNA may be affected by the water environment, time in the water, method for recovery, transport and storage routines of the objects before the objects arrive in the laboratory. The present study evaluated the effect of four storage conditions on the DNA retrieved from bloodstains, touch DNA, fingerprints and hairs, initially deposited on knives, smartphones, packing tapes, duct tapes and garbage bags, and submerged in lake water for three time periods. After retrieval, the objects were stored either through air-drying at room temperature, freezing at -30 °C, in nitrogen gas or in lake water. The results showed that the submersion time strongly influenced the amount and degradation of DNA, especially after the longest submersion time (21 days). A significant variation was observed in success for STR profiling, while mtDNA profiling was less affected by the submersion time interval and storage conditions. This study illustrates that retrieval from water as soon as possible and immediate storage through air-drying or freezing before DNA analysis is beneficial for the outcome of DNA profiling in crime scene investigations.

Clinical and Genomic Predictors of Adverse Events in Newly Diagnosed Glioblastoma.

PURPOSE: Adverse clinical events cause significant morbidity in patients with GBM (GBM). We examined whether genomic alterations were associated with AE (AE) in patients with GBM. EXPERIMENTAL DESIGN: We identified adults with histologically confirmed IDH-wild-type GBM with targeted next-generation sequencing (OncoPanel) at Dana Farber Cancer Institute from 2013 to 2019. Seizure at presentation, lymphopenia, thromboembolic events, pseudoprogression, and early progression (within 6 months of diagnosis) were identified as AE. The biologic function of genetic variants was categorized as loss-of-function (LoF), no change in function, or gain-of-function (GoF) using a somatic tumor mutation knowledge base (OncoKB) and consensus protein function predictions. Associations between functional genomic alterations and AE were examined using univariate logistic regressions and multivariable regressions adjusted for additional clinical predictors. RESULTS: Our study included 470 patients diagnosed with GBM who met the study criteria. We focused on 105 genes that had sequencing data available for ≥ 90% of the patients and were altered in ≥10% of the cohort. Following false-discovery rate (FDR) correction and multivariable adjustment, the TP53, RB1, IGF1R, and DIS3 LoF alterations were associated with lower odds of seizures, while EGFR, SMARCA4, GNA11, BRD4, and TCF3 GoF and SETD2 LoF alterations were associated with higher odds of seizures. For all other AE of interest, no significant associations were found with genomic alterations following FDR correction. CONCLUSIONS: Genomic biomarkers based on functional variant analysis of a routine clinical panel may help identify AE in GBM, particularly seizures. Identifying these risk factors could improve the management of patients through better supportive care and consideration of prophylactic therapies.

Complete Mitochondrial DNA Genome Variation in the Swedish Population.

The development of complete mitochondrial genome (mitogenome) reference data for inclusion in publicly available population databases is currently underway, and the generation of more high-quality mitogenomes will only enhance the statistical power of this forensically useful locus. To characterize mitogenome variation in Sweden, the mitochondrial DNA (mtDNA) reads from the SweGen whole genome sequencing (WGS) dataset were analyzed. To overcome the interference from low-frequency nuclear mtDNA segments (NUMTs), a 10% variant frequency threshold was applied for the analysis. In total, 934 forensic-quality mitogenome haplotypes were characterized. Almost 45% of the SweGen haplotypes belonged to haplogroup H. Nearly all mitogenome haplotypes (99.1%) were assigned to European haplogroups, which was expected based on previous mtDNA studies of the Swedish population. There were signature northern Swedish and Finnish haplogroups observed in the dataset (e.g., U5b1, W1a), consistent with the nuclear DNA analyses of the SweGen data. The complete mitogenome analysis resulted in high haplotype diversity (0.9996) with a random match probability of 0.15%. Overall, the SweGen mitogenomes provide a large mtDNA reference dataset for the Swedish population and also contribute to the effort to estimate global mitogenome haplotype frequencies.

Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile.

Clostridioides difficile produces toxins that damage the colonic epithelium, causing colitis. Variation in disease severity is poorly understood and has been attributed to host factors and virulence differences between C. difficile strains. We test 23 epidemic ST1 C. difficile clinical isolates for their virulence in mice. All isolates encode a complete Tcd pathogenicity locus and achieve similar colonization densities. However, disease severity varies from lethal to avirulent infections. Genomic analysis of avirulent isolates reveals a 69-bp deletion in the cdtR gene, which encodes a response regulator for binary toxin expression. Deleting the 69-bp sequence in virulent R20291 strain renders it avirulent in mice with reduced toxin gene transcription. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile isolates without reducing colonization and persistence. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.

Forensic prediction of sex, age, height, body mass index, hip-to-waist ratio, smoking status and lipid lowering drugs using epigenetic markers and plasma proteins.

The prediction of human characteristics from blood using molecular markers would be very helpful in forensic science. Such information can be particularly important in providing investigative leads in police casework from, for example, blood found at crime scenes in cases without a suspect. Here, we investigated the possibilities and limitations of predicting seven phenotypic traits (sex, age, height, body mass index [BMI], hip-to-waist [WTH] ratio, smoking status and lipid-lowering drug use) using either DNA methylation or plasma proteins separately or in combination. We developed a prediction pipeline starting with the prediction of sex followed by sex-specific, stepwise, individual age, sex-specific anthropometric traits and, finally, lifestyle-related traits. Our data revealed that age, sex and smoking status can be accurately predicted from DNA methylation alone, while the use of plasma proteins was highly accurate for prediction of the WTH ratio, and a combined analysis of the best predictions for BMI and lipid-lowering drug use. In unseen individuals, age was predicted with a standard error of 3.3 years for women and 6.5 years for men, while the accuracy in smoking prediction across both men and women was 0.86. In conclusion, we have developed a stepwise approach for the de-novo prediction of individual characteristics from plasma proteins and DNA methylation markers. These models are accurate and may provide valuable information and investigative leads in future forensic casework.

Clostridioides difficile S-Layer Protein A (SlpA) Serves as a General Phage Receptor.

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, buthe lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but available data are still limited. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by ϕCD38-2, ϕCD111, and ϕCD146, which normally infect the parental strain. Complementation with 12 different S-layer cassette types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by ϕCD111 and SLCT-11 enabled infection by ϕCD38-2 and ϕCD146. Of note, the expression of SLCT-1, -6, -8, -9, -10, or -12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain. Also, deletion of the D2 domain within the low-molecular-weight fragment of SlpA was found to abolish infection by ϕCD38-2 and ϕCD146 but not ϕCD111. Altogether, our data suggest that many phages use SlpA as their receptor and, most importantly, that both siphophages and myophages target SlpA despite major differences in their tail structures. Our study therefore represents an important step in understanding the interactions between C. difficile and its phages. IMPORTANCE Phage therapy represents an interesting alternative to treat Clostridioides difficile infections because, contrary to antibiotics, most phages are highly species specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage receptor-binding protein binds to a specific SLCT, the identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.

The DNA-Buster: The evaluation of an alternative DNA recovery approach.

Touch DNA recovery techniques can have limitations, as their effectiveness depends on the substrate on which the DNA of a person of interest can be found. In this study, an in-house dry-vacuuming device, the DNA-Buster, was compared to traditional methods for its DNA recovery performance from items typically examined in forensic casework. The aim was to evaluate whether this dry-vacuuming approach can recover DNA efficiently, potentially complementing the well-established recovery strategies. For this, the performances of swabbing, taping, wet- (M-Vac®) and dry-vacuuming (DNA-Buster) were investigated quantitatively and qualitatively for touch DNA deposited on carpet, cotton sweater, stone, tile and wood. For the sweater, both vacuuming methods outperformed the other collection tools quantitatively. While the highest DNA amounts for the carpet were yielded by swabbing and taping, dry-vacuuming was equally good in reaching full DNA profiles, whereas less complete profiles were observed for the M-Vac®. For stone and tile, swabbing was optimal, whereas dry-vacuuming clearly underperformed for these substrates. Taping was the best recovery method for wood. Despite applying single donor DNA after thoroughly cleaning the items, undesired DNA mixtures were detected for all recovery techniques and all substrates. The overall research findings show first that the novel dry-vacuuming method is suited for DNA recovery from textiles. Secondly, they indicate that more attention should be paid to the substrate-collection dependency to ensure best practices in recovering genetic material in a precise, confident and targeted manner from the variety of forensic casework material.

Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile.

Clostridioides difficile (C. difficile) , a leading cause of nosocomial infection, produces toxins that damage the colonic epithelium and results in colitis that varies from mild to fulminant. Variation in disease severity is poorly understood and has been attributed to host factors (age, immune competence and intestinal microbiome composition) and/or virulence differences between C. difficile strains, with some, such as the epidemic BI/NAP1/027 (MLST1) strain, being associated with greater virulence. We tested 23 MLST1(ST1) C. difficile clinical isolates for virulence in antibiotic-treated C57BL/6 mice. All isolates encoded a complete Tcd pathogenicity locus and achieved similar colonization densities in mice. Disease severity varied, however, with 5 isolates causing lethal infections, 16 isolates causing a range of moderate infections and 2 isolates resulting in no detectable disease. The avirulent ST1 isolates did not cause disease in highly susceptible Myd88 -/- or germ-free mice. Genomic analysis of the avirulent isolates revealed a 69 base-pair deletion in the N-terminus of the cdtR gene, which encodes a response regulator for binary toxin (CDT) expression. Genetic deletion of the 69 base-pair cdtR sequence in the highly virulent ST1 R20291 C. difficile strain rendered it avirulent and reduced toxin gene transcription in cecal contents. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile strain without reducing colonization and persistence in the gut. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.

The Value of Whole-Genome Sequencing for Mitochondrial DNA Population Studies: Strategies and Criteria for Extracting High-Quality Mitogenome Haplotypes.

Whole-genome sequencing (WGS) data present a readily available resource for mitochondrial genome (mitogenome) haplotypes that can be utilized for genetics research including population studies. However, the reconstruction of the mitogenome is complicated by nuclear mitochondrial DNA (mtDNA) segments (NUMTs) that co-align with the mtDNA sequences and mimic authentic heteroplasmy. Two minimum variant detection thresholds, 5% and 10%, were assessed for the ability to produce authentic mitogenome haplotypes from a previously generated WGS dataset. Variants associated with NUMTs were detected in the mtDNA alignments for 91 of 917 (~8%) Swedish samples when the 5% frequency threshold was applied. The 413 observed NUMT variants were predominantly detected in two regions (nps 12,612-13,105 and 16,390-16,527), which were consistent with previously documented NUMTs. The number of NUMT variants was reduced by ~97% (400) using a 10% frequency threshold. Furthermore, the 5% frequency data were inconsistent with a platinum-quality mitogenome dataset with respect to observed heteroplasmy. These analyses illustrate that a 10% variant detection threshold may be necessary to ensure the generation of reliable mitogenome haplotypes from WGS data resources.

Evaluation of Different Cleaning Strategies for Removal of Contaminating DNA Molecules.

Decontamination strategies and their efficiencies are crucial when performing routine forensic analysis, and many factors influence the choice of agent to use. In this study, the effects of ten different cleaning strategies were evaluated to compare their ability to remove contaminating DNA molecules. Cell-free DNA or blood was deposited on three surfaces (plastic, metal, and wood) and decontaminated with various treatments. The quantities of recovered DNA, obtained by swabbing the surfaces after cleaning using the different strategies, was analyzed by real-time PCR. Large differences in the DNA removal efficiencies were observed between different cleaning strategies, as well as between different surfaces. The most efficient cleaning strategies for cell-free DNA were the different sodium hypochlorite solutions and Trigene®, for which a maximum of 0.3% DNA was recovered on all three surfaces. For blood, a maximum of 0.8% of the deposited DNA was recovered after using Virkon® for decontamination. The recoveries after using these cleaning strategies correspond to DNA from only a few cells, out of 60 ng of cell-free DNA or thousands of deposited blood cells.

Clinical utility of targeted next-generation sequencing assay in IDH-wildtype glioblastoma for therapy decision-making.

BACKGROUND: Targeted gene NGS testing is available through many academic institutions and commercial entities and is increasingly incorporated in practice guidelines for glioblastoma (GBM). This single-center retrospective study aimed to evaluate the clinical utility of incorporating NGS results in the management of GBM patients at a clinical trials-focused academic center. METHODS: We identified 1011 consecutive adult patients with pathologically confirmed GBM (IDHwt or IDHmut) who had somatic tumor sequencing (Oncopanel, ~500 cancer gene panel) at DFCI from 2013-2019. Clinical records of all IDHwt GBM patients were reviewed to capture clinical trial enrollment and off-label targeted therapy use based on NGS results. RESULTS: Of the 557 IDHwt GBM patients with sequencing, 182 entered clinical trials at diagnosis (32.7%) and 213 (38.2%) entered after recurrence. Sequencing results for 130 patients (23.3%) were utilized for clinical trial enrollment for either targeted therapy indications (6.9 % upfront and 27.7% at recurrent clinical trials and 3.1% for off-label targeted therapy) or exploratory studies (55.4% upfront and 6.9% recurrent clinical trials). Median overall survival was 20.1 months with no survival difference seen between patients enrolled in clinical trials compared to those who were not, in a posthoc analysis. CONCLUSIONS: While NGS testing has become essential for improved molecular diagnostics, our study illustrates that targeted gene panels remain underutilized for selecting therapy in GBM-IDHwt. Targeted therapy and clinical trial design remain to be improved to help leverage the potential of NGS in clinical care.

Population genomics of the Viking world.

The maritime expansion of Scandinavian populations during the Viking Age (about AD 750-1050) was a far-flung transformation in world history1,2. Here we sequenced the genomes of 442 humans from archaeological sites across Europe and Greenland (to a median depth of about 1×) to understand the global influence of this expansion. We find the Viking period involved gene flow into Scandinavia from the south and east. We observe genetic structure within Scandinavia, with diversity hotspots in the south and restricted gene flow within Scandinavia. We find evidence for a major influx of Danish ancestry into England; a Swedish influx into the Baltic; and Norwegian influx into Ireland, Iceland and Greenland. Additionally, we see substantial ancestry from elsewhere in Europe entering Scandinavia during the Viking Age. Our ancient DNA analysis also revealed that a Viking expedition included close family members. By comparing with modern populations, we find that pigmentation-associated loci have undergone strong population differentiation during the past millennium, and trace positively selected loci-including the lactase-persistence allele of LCT and alleles of ANKA that are associated with the immune response-in detail. We conclude that the Viking diaspora was characterized by substantial transregional engagement: distinct populations influenced the genomic makeup of different regions of Europe, and Scandinavia experienced increased contact with the rest of the continent.

Impact of the sequencing method on the detection and interpretation of mitochondrial DNA length heteroplasmy.

Advancements in sequencing technologies allow for rapid and efficient analysis of mitochondrial DNA (mtDNA) in forensic laboratories, which is particularly beneficial for specimens with limited nuclear DNA. Next generation sequencing (NGS) offers higher throughput and sensitivity over traditional Sanger-type sequencing (STS) as well as the ability to quantitatively analyze the data. Changes in sample preparation, sequencing method and analysis required for NGS may alter the mtDNA haplotypes compared to previously generated STS data. Thus, the present study aimed to characterize the impact of different sequencing workflows on the detection and interpretation of length heteroplasmy (LHP), a particularly complicated aspect of mtDNA analysis. Whole mtDNA genome (mitogenome) data were generated for 16 high-quality samples using well-established Illumina and Ion methods, and the NGS data were compared to previously-generated STS mtDNA control region data. Although the mitogenome haplotypes were concordant with the exception of length and low-level variants (<30 % variant frequency), LHP in the hypervariable segment (HVS) polycytosine regions (C-tracts) differed across sequencing methods. Consistent with previous studies, LHP in HVS1 was observed in samples with nine or more consecutive cytosines (Cs) and eight Cs in the HVS2 region in the STS data. The Illumina data produced a similar pattern of LHP as the STS data, whereas the Ion data were noticeably different. More complex LHP (i.e. more length molecules) was observed in the Ion data, as length variation occurred in multiple homopolymer stretches within the targeted HVS regions. Further, the STS dominant or major molecule (MM) differed from the Ion MM in 11 (37 %) of the 30 regions evaluated and six instances (20 %) in Illumina data. This is of particular interest, as the MM is used by many forensic laboratories to report the HVS C-tract in the mtDNA haplotype. In general, the STS MMs were longer than the Illumina MMs, while the Ion MMs were the shortest. The higher rate of homopolymer indels in Ion data likely contributed to these differences. Supplemental analysis with alternative approaches demonstrated that the LHP pattern may also be altered by the bioinformatic tool and workflow used for data interpretation. The broader application of NGS in forensic laboratories will undoubtedly result in the use of varying sample preparation and sequencing methods. Based on these findings, minor LHP differences are expected across sequencing workflows, and it will be important that C-tract indels continue to be ignored for forensic queries and comparisons.

Mitochondrial DNA analysis of a Viking age mass grave in Sweden.

In 1998, a Viking Age mass grave was discovered and excavated at St. Laurence´s churchyard in Sigtuna, Sweden. The excavated bones underwent osteoarchaeological analysis and were assigned to at least 19 individuals. Eleven skeletons showed sharp force trauma from bladed weapons. Mass graves are an unusual finding from this time period, making the burial context extraordinary. To investigate a possible maternal kinship among the individuals, bones and teeth from the skeletal remains were selected for mitochondrial DNA (mtDNA) analysis. Sanger sequencing of short stretches of the hypervariable segments I and II (HVS-I and HVS-II) was performed. A subset of the samples was also analysed by massively parallel sequencing analysis (MPS) of the entire mtDNA genome using the Precision ID mtDNA Whole Genome Panel. A total of 15 unique and three shared mtDNA profiles were obtained. Based on a combination of genetic and archaeological data, we conclude that a minimum of 20 individuals was buried in the mass grave. The majority of the individuals were not maternally related. However, two possible pairs of siblings or mother-child relationships were identified. All individuals were assigned to West Eurasian haplogroups, with a predominance of haplogroup H. Although the remains showed an advanced level of DNA degradation, the combined use of Sanger sequencing and MPS with the Precision ID mtDNA Whole Genome Panel revealed at least partial mtDNA data for all samples.

Evaluation of the precision ID whole MtDNA genome panel for forensic analyses.

Mitochondrial DNA (mtDNA) amplification and Massively Parallel Sequencing (MPS) using an early access version of the Precision ID Whole MtDNA Genome Panel (Thermo Fisher Scientific) and the Ion Personal Genome Machine (PGM) were evaluated using 15 forensically relevant samples. Samples were selected to represent typical forensic specimens for mtDNA analysis including hairs, hair shafts, swabs and ancient solid tissue samples (bones and teeth) that were stored in the freezer for up to several years after having been typed with conventional Sanger-type Sequencing and Capillary Electrophoresis. The MPS haplotypes confirmed the earlier results in all samples and provided additional sequence information that improved discrimination power and haplogroup estimation. The results raised the appetite for further experiments to validate and apply the new technology in forensic practice.

Results of a collaborative study on DNA identification of aged bone samples.

AIM: A collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. METHODS: Eighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. RESULTS: Although the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. CONCLUSION: The laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized.

Multiplex pyrosequencing of InDel markers for forensic DNA analysis.

The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator® DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator® DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof-of-principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost-effective alternative for some applications.

Origin of the U87MG glioma cell line: Good news and bad news.

Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.

Comparison of DNA polymerases for improved forensic analysis of challenging samples.

Inhibitors of polymerase chain reaction (PCR) amplification often present a challenge in forensic investigations of e.g., terrorism, missing persons, sexual assaults and other criminal cases. Such inhibitors may be counteracted by dilution of the DNA extract, using different additives, and selecting an inhibitory resistant DNA polymerase. Additionally, DNA in forensic samples is often present in limited amounts and degraded, requiring special analyses of short nuclear targets or mitochondrial DNA. The present study evaluated the enzymes AmpliTaq Gold, HotStarTaq Plus, KAPA3G Plant, and KAPA2G Robust, with regard to their ability to overcome inhibitory effects. Our data showed that diluting the extracts and adding bovine serum albumin may increase the yield of the PCR product. However, the largest impact was observed when alternative enzymes were utilized, instead of the commonly used AmpliTaq Gold. KAPA2G Robust presented the highest amplification efficiency in the presence of the inhibitor ammonium nitrate. Moreover, the KAPA3G Plant enzyme had the highest efficiency in amplifying degraded DNA from old buried bone material. KAPA3G Plant and KAPA2G Robust may thus be useful for counteracting inhibitors and improving the analysis of challenging samples.