Targeting ferroptosis in ovarian cancer: Novel strategies to overcome chemotherapy resistance.

AIMS: This review investigates the role of ferroptosis in combating chemotherapy resistance in ovarian cancer, with a focus on its underlying mechanisms and therapeutic implications. MAIN METHODS: A database search was conducted up to December 2023 using PubMed, Scopus, Google Scholar, Web of Science, and the Cochrane Library. The keywords "ovarian cancer," "ferroptosis," "cisplatin," and "cisplatin resistance" were employed. We included studies that offered original data on the application of ferroptosis in platinum-based chemotherapy, focusing on both in-vitro and in-vivo research models. KEY FINDINGS: Our review reveals that ferroptosis significantly influences drug resistance in ovarian cancer. It investigates the existing studies to understand the role of ferroptosis in platinum resistance and explores its underlying mechanisms and assesses potential therapeutic strategies that uses ferroptosis to improve outcomes. The findings underscore the importance of ferroptosis in enhancing the effectiveness of platinum-based treatments and improving patient prognosis. SIGNIFICANCE: The potential of ferroptosis induction to develop novel therapeutic strategies against ovarian cancer, especially in cisplatin-resistant cases, is promising. The preliminary nature of these findings highlights the necessity for further research to bring these insights into clinical practice. This would not only improve treatment outcomes and prognosis but also encourage ongoing studies into ferroptosis as a viable therapeutic approach.

Minerals and the Menstrual Cycle: Impacts on Ovulation and Endometrial Health.

The role of minerals in female fertility, particularly in relation to the menstrual cycle, presents a complex area of study that underscores the interplay between nutrition and reproductive health. This narrative review aims to elucidate the impacts of minerals on key aspects of the reproductive system: hormonal regulation, ovarian function and ovulation, endometrial health, and oxidative stress. Despite the attention given to specific micronutrients in relation to reproductive disorders, there is a noticeable absence of a comprehensive review focusing on the impact of minerals throughout the menstrual cycle on female fertility. This narrative review aims to address this gap by examining the influence of minerals on reproductive health. Each mineral's contribution is explored in detail to provide a clearer picture of its importance in supporting female fertility. This comprehensive analysis not only enhances our knowledge of reproductive health but also offers clinicians valuable insights into potential therapeutic strategies and the recommended intake of minerals to promote female reproductive well-being, considering the menstrual cycle. This review stands as the first to offer such a detailed examination of minerals in the context of the menstrual cycle, aiming to elevate the understanding of their critical role in female fertility and reproductive health.

Immune Response Associated Gene Signatures in Aortic Dissection Compared to Aortic Aneurysm.

BACKGROUND: Thoracic aortic dissections (TAD) are life-threatening events mostly requiring immediate surgical treatment. Although dissections mainly occur independently of thoracic aortic aneurysms (TAA), both share a high comorbidity. There are several indications for an involvement of the immune system in the development of TAD, just as in TAA. Nevertheless, specific disease-relevant genes, biomolecular processes, and immune-specific phenotypes remain unknown. METHODS: RNA from isolated aortic smooth muscle cells from TAD (n = 4), TAA (n = 3), and control patients were analyzed using microarray-based technologies. Additionally, three publicly available bulk RNA-seq studies of TAD (n = 23) and controls (n = 17) and one single-cell RNA-seq study of TAA (n = 8) and controls (n = 3) were analyzed. Differentially expressed genes were identified and used to identify affected pathways in TAD. Five selected genes were validated by quantitative real-time polymerase chain reaction (PCR). RESULTS: We identified 37 genes that were significantly dysregulated in at least three TAD studies-24 of them were not shown to be associated with TAD, yet. Gene ontology analysis showed that immune response was significantly affected. Five of the genes (CCL2, RNASE2, HAVCR2, CXCL8, and IL6R) were revealed as core genes that affect immune response in TAD. We compared the gene expression of those genes to TAA and found that CXCL8, IL6R, and potentially also CCL2 were upregulated in TAD. CONCLUSIONS: The identified immune-related genes showed TAD-specificity, independent of possible pre-existing comorbidities like TAA. So, these genes represent potential biomarkers and therapeutic targets linked to the immune response in acute TAD. Additionally, we identified a set of differentially expressed genes that represents a resource for further studies.

Exploring the Micro-Mosaic Landscape of FGFR3 Mutations in the Ageing Male Germline and Their Potential Implications in Meiotic Differentiation.

Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants-c.1138G>A (p.G380R) and c.1948A>G (p.K650E)-which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor's age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline.

Effects of age on the diagnostic value of the soluble fms-like tyrosine kinase-1/ placental growth factor ratio in preeclampsia: a retrospective cohort study.

OBJECTIVE: Measurement of the ratio between soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) supports the diagnosis of preeclampsia. sFlt-1/PlGF ratios of at least 85 and at least 110 have previously been suggested for diagnosis of early-onset and late-onset preeclampsia, respectively. However, angiogenic and antiangiogenic factors change throughout the process of aging, potentially influencing preeclampsia diagnosis. In this study, we therefore evaluated in detail the effect of maternal age on sFlt-1/PlGF ratios. METHODS: A total of 2775 pregnant female patients were included in this retrospective cohort study, spread across three maternal age groups: 18-25 years, 26-35 years, and more than 35 years at delivery. Receiver-operating characteristic (ROC) curve analysis was employed to evaluate sFlt-1/PlGF ratio cutoffs for use in preeclampsia diagnosis. RESULTS: Controls (2462 pregnant women) showed a significant difference in sFlt-1/PlGF ratios between the youngest and oldest age groups, which resulted in differences in the best-performing sFlt-1/PlGF ratio cutoffs: optimized cutoffs were 143.4 (52.9%, 98.2%), 8.6 (84.4%, 75.3%), and 22.9 (78.6%, 82.3%) in early-onset preeclampsia, and 46.4 (67.5%, 81.5%), 40.8 (77.3%, 73%), and 44.1 (65.1%, 74.5%) in late-onset preeclampsia in age groups, 1, 2, and 3, respectively. CONCLUSION: sFlt-1/PlGF ratios change with maternal age, which has important clinical implications for their use in the diagnosis of preeclampsia: Better differentiated sFlt-1/PlGF cutoffs should be used that take maternal and gestational age into account.

Noncanonical atherosclerosis as the driving force in tricuspid aortic valve associated aneurysms - A trace collection.

Pathogenic mechanisms in degenerative thoracic aortic aneurysms (TAA) are still unclear. There is an ongoing debate about whether TAAs are caused by uniform or distinct processes, which would obviously have a major impact on future treatment strategies. Clearly, the ultimate outcome of TAA subgroups associated with a tricuspid aortic valve (TAV) or a bicuspid aortic valve (BAV) is the same, namely a TAA. Based on results from our own and others' studies, we decided to compare the different TAAs (TAV and BAV) and controls using a broad array of analyses, i.e., metabolomic analyses, gene expression profiling, protein expression analyses, histological characterization, and matrix-assisted laser desorption ionization imaging. Central findings of the present study are the presence of noncanonical atherosclerosis, pathological accumulation of macrophages, and disturbances of lipid metabolism in the aortic media. Moreover, we have also found that lipid metabolism is impaired systemically. Importantly, all of the above-described phenotypes are characteristic for TAV-TAA only, and not for BAV-TAA. In summary, our results suggest different modes of pathogenesis in TAV- and BAV-associated aneurysms. Intimal atherosclerotic changes play a more central role in TAV-TAA formation than previously thought, particularly as the observed alterations do not follow classical patterns. Atherosclerotic alterations are not limited to the intima but also affect and alter the TAV-TAA media. Further studies are needed to i) clarify patho-relevant intima-media interconnections, ii) define the origin of the systemic alteration of lipid metabolism, and iii) to define valid biomarkers for early diagnosis, disease progression, and successful treatments in TAV-TAAs.

Biomarker screening in preeclampsia: an RNA-sequencing approach based on data from multiple studies.

OBJECTIVE: Biomarkers have become important in the prognosis and diagnosis of various diseases. High-throughput methods, such as RNA sequencing facilitate the detection of differentially expressed genes (DEGs), hence potential biomarker candidates. Individual studies suggest long lists of DEGs, hampering the identification of clinically relevant ones. Concerning preeclampsia - a major obstetric burden with high risk for adverse maternal and/or neonatal outcomes - limitations in diagnosis and prediction are still important issues. We, therefore, developed a workflow to facilitate the screening for biomarkers. METHODS: On the basis of the tool DESeq2, a comprehensive workflow for identifying DEGs was established, analyzing data from several publicly available RNA-sequencing studies. We applied it to four RNA-sequencing datasets (one blood, three placenta) analyzing patients with preeclampsia and normotensive controls. We compared our results with other published approaches and evaluated their performance. RESULTS: We identified 110 genes that are dysregulated in preeclampsia, observed in at least three of the studies analyzed, six even in all four studies. These included FLT-1, TREM-1, and FN1, which either represent established biomarkers at protein level, or promising candidates based on recent studies. For comparison, using a published meta-analysis approach, 5240 DEGs were obtained. CONCLUSION: This study presents a data analysis workflow for preeclampsia biomarker screening, capable of identifying promising biomarker candidates, while drastically reducing the numbers of candidates. Moreover, we were also able to confirm its performance for heart failure. This approach can be applied to additional diseases for biomarker identification, and the set of DEGs identified in preeclampsia represents a resource for further studies.

Advanced age increases frequencies of de novo mitochondrial mutations in macaque oocytes and somatic tissues.

Mutations in mitochondrial DNA (mtDNA) contribute to multiple diseases. However, how new mtDNA mutations arise and accumulate with age remains understudied because of the high error rates of current sequencing technologies. Duplex sequencing reduces error rates by several orders of magnitude via independently tagging and analyzing each of the two template DNA strands. Here, using duplex sequencing, we obtained high-quality mtDNA sequences for somatic tissues (liver and skeletal muscle) and single oocytes of 30 unrelated rhesus macaques, from 1 to 23 y of age. Sequencing single oocytes minimized effects of natural selection on germline mutations. In total, we identified 17,637 tissue-specific de novo mutations. Their frequency increased ∼3.5-fold in liver and ∼2.8-fold in muscle over the ∼20 y assessed. Mutation frequency in oocytes increased ∼2.5-fold until the age of 9 y, but did not increase after that, suggesting that oocytes of older animals maintain the quality of their mtDNA. We found the light-strand origin of replication (OriL) to be a hotspot for mutation accumulation with aging in liver. Indeed, the 33-nucleotide-long OriL harbored 12 variant hotspots, 10 of which likely disrupt its hairpin structure and affect replication efficiency. Moreover, in somatic tissues, protein-coding variants were subject to positive selection (potentially mitigating toxic effects of mitochondrial activity), the strength of which increased with the number of macaques harboring variants. Our work illuminates the origins and accumulation of somatic and germline mtDNA mutations with aging in primates and has implications for delayed reproduction in modern human societies.

Discovery of an unusually high number of de novo mutations in sperm of older men using duplex sequencing.

De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultradeep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ∼4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant allele frequencies (VAFs) of 10-4 to 10-5, with an overall mutation frequency of the region of ∼6 × 10-7 Some of the substitutions are recurrent and are found at a higher VAF in older donors than in younger ones or are found exclusively in older donors. Also, older donors harbor more mutations associated with congenital disorders. Other mutations are present in both age groups, suggesting that these might result from a different mechanism (e.g., postzygotic mosaicism). We also observe that independent of age, the frequency and deleteriousness of the mutational spectra are more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain of function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

Age-related accumulation of de novo mitochondrial mutations in mammalian oocytes and somatic tissues.

Mutations create genetic variation for other evolutionary forces to operate on and cause numerous genetic diseases. Nevertheless, how de novo mutations arise remains poorly understood. Progress in the area is hindered by the fact that error rates of conventional sequencing technologies (1 in 100 or 1,000 base pairs) are several orders of magnitude higher than de novo mutation rates (1 in 10,000,000 or 100,000,000 base pairs per generation). Moreover, previous analyses of germline de novo mutations examined pedigrees (and not germ cells) and thus were likely affected by selection. Here, we applied highly accurate duplex sequencing to detect low-frequency, de novo mutations in mitochondrial DNA (mtDNA) directly from oocytes and from somatic tissues (brain and muscle) of 36 mice from two independent pedigrees. We found mtDNA mutation frequencies 2- to 3-fold higher in 10-month-old than in 1-month-old mice, demonstrating mutation accumulation during the period of only 9 mo. Mutation frequencies and patterns differed between germline and somatic tissues and among mtDNA regions, suggestive of distinct mutagenesis mechanisms. Additionally, we discovered a more pronounced genetic drift of mitochondrial genetic variants in the germline of older versus younger mice, arguing for mtDNA turnover during oocyte meiotic arrest. Our study deciphered for the first time the intricacies of germline de novo mutagenesis using duplex sequencing directly in oocytes, which provided unprecedented resolution and minimized selection effects present in pedigree studies. Moreover, our work provides important information about the origins and accumulation of mutations with aging/maturation and has implications for delayed reproduction in modern human societies. Furthermore, the duplex sequencing method we optimized for single cells opens avenues for investigating low-frequency mutations in other studies.

Family reunion via error correction: an efficient analysis of duplex sequencing data.

BACKGROUND: Duplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies. Its power comes from pooling together multiple descendants of both strands of original DNA molecules, which allows distinguishing true nucleotide substitutions from PCR amplification and sequencing artifacts. This strategy comes at a cost-sequencing the same molecule multiple times increases dynamic range but significantly diminishes coverage, making whole genome duplex sequencing prohibitively expensive. Furthermore, every duplex experiment produces a substantial proportion of singleton reads that cannot be used in the analysis and are thrown away. RESULTS: In this paper we demonstrate that a significant fraction of these reads contains PCR or sequencing errors within duplex tags. Correction of such errors allows "reuniting" these reads with their respective families increasing the output of the method and making it more cost effective. CONCLUSIONS: We combine an error correction strategy with a number of algorithmic improvements in a new version of the duplex analysis software, Du Novo 2.0. It is written in Python, C, AWK, and Bash. It is open source and readily available through Galaxy, Bioconda, and Github: https://github.com/galaxyproject/dunovo.

Pronounced somatic bottleneck in mitochondrial DNA of human hair.

Heteroplasmy is the presence of variable mitochondrial DNA (mtDNA) within the same individual. The dynamics of heteroplasmy allele frequency among tissues of the human body is not well understood. Here, we measured allele frequency at heteroplasmic sites in two to eight hairs from each of 11 humans using next-generation sequencing. We observed a high variance in heteroplasmic allele frequency among separate hairs from the same individual-much higher than that for blood and cheek tissues. Our population genetic modelling estimated the somatic bottleneck during embryonic follicle development of separate hairs to be only 11.06 (95% confidence interval 0.6-34.0) mtDNA segregating units. This bottleneck is much more drastic than somatic bottlenecks for blood and cheek tissues (136 and 458 units, respectively), as well as more drastic than, or comparable to, the germline bottleneck (equal to 25-32 or 7-10 units, depending on the study). We demonstrated that hair undergoes additional genetic drift before and after the divergence of mtDNA lineages of individual hair follicles. Additionally, we showed a positive correlation between donor's age and variance in heteroplasmy allele frequency in hair. These findings have important implications for forensics and for our understanding of mtDNA dynamics in the human body. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Bottleneck and selection in the germline and maternal age influence transmission of mitochondrial DNA in human pedigrees.

Heteroplasmy-the presence of multiple mitochondrial DNA (mtDNA) haplotypes in an individual-can lead to numerous mitochondrial diseases. The presentation of such diseases depends on the frequency of the heteroplasmic variant in tissues, which, in turn, depends on the dynamics of mtDNA transmissions during germline and somatic development. Thus, understanding and predicting these dynamics between generations and within individuals is medically relevant. Here, we study patterns of heteroplasmy in 2 tissues from each of 345 humans in 96 multigenerational families, each with, at least, 2 siblings (a total of 249 mother-child transmissions). This experimental design has allowed us to estimate the timing of mtDNA mutations, drift, and selection with unprecedented precision. Our results are remarkably concordant between 2 complementary population-genetic approaches. We find evidence for a severe germline bottleneck (7-10 mtDNA segregating units) that occurs independently in different oocyte lineages from the same mother, while somatic bottlenecks are less severe. We demonstrate that divergence between mother and offspring increases with the mother's age at childbirth, likely due to continued drift of heteroplasmy frequencies in oocytes under meiotic arrest. We show that this period is also accompanied by mutation accumulation leading to more de novo mutations in children born to older mothers. We show that heteroplasmic variants at intermediate frequencies can segregate for many generations in the human population, despite the strong germline bottleneck. We show that selection acts during germline development to keep the frequency of putatively deleterious variants from rising. Our findings have important applications for clinical genetics and genetic counseling.

A High-Resolution View of Adaptive Event Dynamics in a Plasmid.

Coadaptation between bacterial hosts and plasmids frequently results in adaptive changes restricted exclusively to host genome leaving plasmids unchanged. To better understand this remarkable stability, we transformed naïve Escherichia coli cells with a plasmid carrying an antibiotic-resistance gene and forced them to adapt in a turbidostat environment. We then drew population samples at regular intervals and subjected them to duplex sequencing-a technique specifically designed for identification of low-frequency mutations. Variants at ten sites implicated in plasmid copy number control emerged almost immediately, tracked consistently across the experiment's time points, and faded below detectable frequencies toward the end. This variation crash coincided with the emergence of mutations on the host chromosome. Mathematical modeling of trajectories for adaptive changes affecting plasmid copy number showed that such mutations cannot readily fix or even reach appreciable frequencies. We conclude that there is a strong selection against alterations of copy number even if it can provide a degree of growth advantage. This incentive is likely rooted in the complex interplay between mutated and wild-type plasmids constrained within a single cell and underscores the importance of understanding of intracellular plasmid variability.

High Satellite Repeat Turnover in Great Apes Studied with Short- and Long-Read Technologies.

Satellite repeats are a structural component of centromeres and telomeres, and in some instances, their divergence is known to drive speciation. Due to their highly repetitive nature, satellite sequences have been understudied and underrepresented in genome assemblies. To investigate their turnover in great apes, we studied satellite repeats of unit sizes up to 50 bp in human, chimpanzee, bonobo, gorilla, and Sumatran and Bornean orangutans, using unassembled short and long sequencing reads. The density of satellite repeats, as identified from accurate short reads (Illumina), varied greatly among great ape genomes. These were dominated by a handful of abundant repeated motifs, frequently shared among species, which formed two groups: 1) the (AATGG)n repeat (critical for heat shock response) and its derivatives; and 2) subtelomeric 32-mers involved in telomeric metabolism. Using the densities of abundant repeats, individuals could be classified into species. However, clustering did not reproduce the accepted species phylogeny, suggesting rapid repeat evolution. Several abundant repeats were enriched in males versus females; using Y chromosome assemblies or Fluorescent In Situ Hybridization, we validated their location on the Y. Finally, applying a novel computational tool, we identified many satellite repeats completely embedded within long Oxford Nanopore and Pacific Biosciences reads. Such repeats were up to 59 kb in length and consisted of perfect repeats interspersed with other similar sequences. Our results based on sequencing reads generated with three different technologies provide the first detailed characterization of great ape satellite repeats, and open new avenues for exploring their functions.

The impact of poly-A microsatellite heterologies in meiotic recombination.

Meiotic recombination has strong, but poorly understood effects on short tandem repeat (STR) instability. Here, we screened thousands of single recombinant products with sperm typing to characterize the role of polymorphic poly-A repeats at a human recombination hotspot in terms of hotspot activity and STR evolution. We show that the length asymmetry between heterozygous poly-A's strongly influences the recombination outcome: a heterology of 10 A's (9A/19A) reduces the number of crossovers and elevates the frequency of non-crossovers, complex recombination products, and long conversion tracts. Moreover, the length of the heterology also influences the STR transmission during meiotic repair with a strong and significant insertion bias for the short heterology (6A/7A) and a deletion bias for the long heterology (9A/19A). In spite of this opposing insertion-/deletion-biased gene conversion, we find that poly-A's are enriched at human recombination hotspots that could have important consequences in hotspot activation.

Haplotyping of Heterozygous SNPs in Genomic DNA Using Long-Range PCR.

To study meiotic recombination products, cis- or trans-association of disease polymorphisms, or allele-specific expression patterns, it is necessary to phase heterozygous polymorphisms separated by several kilobases. Haplotyping using long-range polymerase chain reaction (PCR) is a powerful, cost-effective method to directly obtain the phase of multiple heterozygous sites with standard laboratory equipment in a handful of loci for many samples. The method is based on the amplification of large genomic DNA regions (up to ~40 kb) with a reaction mixture that combines a proofreading polymerase with allele-specific primer pairs that preferentially amplify matched templates. The analysis of two heterozygous SNPs requires four reactions, each containing one of the four possible allele-specific primer combinations (two forward and two reverse primers), with the mismatches occurring at the 3' ends of the primers. The two correct primer combinations will more efficiently elongate the matching alleles than the alternative alleles, and the difference in amplification efficiency can be monitored with real-time PCR.

High-Throughput Genotyping with TaqMan Allelic Discrimination and Allele-Specific Genotyping Assays.

Real-time PCR-based genotyping methods, such as TaqMan allelic discrimination assays and allele-specific genotyping, are particularly useful when screening a handful of single nucleotide polymorphisms in hundreds of samples; either derived from different individuals, tissues, or pre-amplified DNA. Although real-time PCR-based methods such as TaqMan are well-established, alternative methods, like allele-specific genotyping, are powerful alternatives, especially for genotyping short tandem repeat (STR) length polymorphisms. Here, we describe all relevant aspects when developing an assay for a new SNP or STR using either TaqMan or allele-specific genotyping, respectively, such as primer and probe design, optimization of reaction conditions, the experimental procedure for typing hundreds of samples, and finally the data evaluation. Our goal is to provide a guideline for developing genotyping assays using these two approaches that render reliable and reproducible genotype calls involving minimal optimization.

Streamlined analysis of duplex sequencing data with Du Novo.

Duplex sequencing was originally developed to detect rare nucleotide polymorphisms normally obscured by the noise of high-throughput sequencing. Here we describe a new, streamlined, reference-free approach for the analysis of duplex sequencing data. We show the approach performs well on simulated data and precisely reproduces previously published results and apply it to a newly produced dataset, enabling us to type low-frequency variants in human mitochondrial DNA. Finally, we provide all necessary tools as stand-alone components as well as integrate them into the Galaxy platform. All analyses performed in this manuscript can be repeated exactly as described at http://usegalaxy.org/duplex .

Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications.

The need in cancer research or evolutionary biology to detect rare mutations or variants present at very low frequencies (<10-5) poses an increasing demand on lowering the detection limits of available methods. Here we demonstrated that amplifiable DNA lesions introduce important error sources in ultrasensitive technologies such as single molecule PCR (smPCR) applications (e.g. droplet-digital PCR), or next-generation sequencing (NGS) based methods. Using templates with known amplifiable lesions (8-oxoguanine, deaminated 5-methylcytosine, uracil, and DNA heteroduplexes), we assessed with smPCR and duplex sequencing that templates with these lesions were amplified very efficiently by proofreading polymerases (except uracil), leading to G->T, and to a lesser extent, to unreported G->C substitutions at 8-oxoguanine lesions, and C->T transitions in amplified uracil containing templates. Long heat incubations common in many DNA extraction protocols significantly increased the number of G->T substitutions. Moreover, in ∼50-80% smPCR reactions we observed the random amplification preference of only one of both DNA strands explaining the known 'PCR jackpot effect', with the result that a lesion became indistinguishable from a true mutation or variant. Finally, we showed that artifactual mutations derived from uracil and 8-oxoguanine could be significantly reduced by DNA repair enzymes.