Repeated DNA Sequences Research Articles

Dysfunction of Telomeric Cdc13-Stn1-Ten1 Simultaneously Activates DNA Damage and Spindle Checkpoints.

Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1, coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1-sz, stn1-sz and cdc13-sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1-sz, stn1-sz and cdc13-sz mutants.

Phenotypic and Molecular-Markers-Based Assessment of Jamun (Syzygium cumini) Genotypes from Pakistan

Jamun plant displays enormous diversity throughout Pakistan, which necessitates its screening, evaluation, and validation to document elite genotypes having better traits for the benefit of the fruit industry and farmers. Surveys were made in natural Jamun habitats across Punjab, Pakistan, and genotypes were marked based on visual diversity of trees and fruits. In total, 60 Jamun genotypes were selected for characterization based on phenotypic and genetic markers. Phenotypic characters related to trees, leaf, and flower along with fruit qualitative traits were assessed in situ. Results revealed significant diversity with high (>25%) coefficient of variance values and the first two components of correspondence analysis exhibited 41.71% variation among genotypes. A strong association was observed among traits like upright tree and round fruit shape (0.74), bluish-colored fruit and pinkish pulp (0.85), and elliptic-shaped fruit with low fruit waxiness (−0.72). Leaves of phenotypically characterized plants were brought to Wheat Biotechnology Lab., University of Agriculture, Faisalabad, Pakistan, where Jamun genotypes were investigated genetically using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. A total of 132 bands were scored, of which 108 were polymorphic, corresponding to almost 81% polymorphism among collected genotypes. High polymorphism information content values were observed against RAPD (0.389) and ISSR (0.457) markers. Genotypes were compared in relation to genetic markers, which exhibited that almost 86% of genetic variability was attributed to differences among accessions, while 14% of variation was due to differences between collections of different areas. Findings of this study confirmed wide phenotypic and genetic distinctness of Jamun in Pakistan that can aid breeders for marker-assisted selection and germplasm enhancement for future crop improvement programs.

Association of hTERT expression, Her2Neu, estrogen receptors, progesterone receptors, with telomere length before and at the end of treatment in breast cancer patients.

Breast cancer shows significant clinical, morphologic, and molecular variation. Telomeres are nucleoprotein complexes composed of hexanucleotide repeat DNA sequence, TTAGGG, and numerous telomere-associated proteins. The maintenance of telomere length is carried out by a ribonucleoprotein called telomerase, which consists of two main components: a catalytic subunit called hTERT (human telomerase reverse transcriptase) and an RNA template called hTR (human telomerase RNA). The importance of evaluating hTERT expression lies in its potential therapeutic application, being an attractive target due to its almost non-existent expression in normal somatic cells. It is also expected that the anti-neoplastic effect would appear earlier in neoplastic cells with shorter telomeres. Additionally, a significant relationship has been observed between Her2-Neu overexpression and Her2-Neu positivity, which could suggest new combined therapies.The aim of this study was to detect the expression of hTERT, estrogen receptor (ER), progesterone receptor (PR), and HER2-Neu in neoplastic breast tissue embedded in paraffin before treatment and to investigate the relationship between them and with baseline and post-treatment telomere length, as well as with various clinicopathological parameters. A cross-sectional-correlational, 21 women diagnosed with breast cancer at the Oncology Service of the High Specialty Medical Unit No. 1 of Bajio of the Mexican Institute of Social Security. The study complies with the Helsinki Declaration and was approved by the Institutional Ethical Committee of the Mexican Institute of Social Security (R-2019-1001-127). A peripheral blood sample was obtained before oncological treatment and at the end of oncological treatment for the measurement of telomere length by extracting DNA from leukocytes, was performed by the quantitative polymerase chain reaction (PCR) method described by Cawthon. Tumor samples were collected from each patient at the oncology department for immunohistochemical determination of biomarker expression (ER, PR, Her2/neu) and hTERT. Of the 21 cases included in the study, the median age was 57.57 years. Eighteen cases were classified as invasive ductal carcinoma NOS (85.71%), 10 were histologic grade 2 (47.61%), 16 cases were hormone receptor positive (76.19%), 7 were Her2Neu positive (33.33%), and only 2 cases were triple negative (9.52%). Positive hTERT expression was detected in 11 cases (52.38%) and was negative in the remaining cases. A significant association was identified between hTERT-positive cases and Her2-Neu positive cases (p = 0.04). Baseline and post-treatment telomere lengths showed a significant difference using the non-parametric Wilcoxon t-test (p = 0.002). In hTERT-positive cases, there was significant telomere shortening at the end of oncological treatment (6.14 ± 1.54 vs. 4.75 ± 1.96 Kb, p = 0.007). Positive hTERT immunostaining cases were associated with poor prognostic factors, such as Her2-Neu overexpression and post-treatment telomere shortening. In the future, hTERT immunostaining could be used to select patients for therapies with antagonistic effects on hTERT, as well as in the selection of more appropriate chemotherapy regimens for patients who express it.

Pharmacotherapeutic Considerations on Telomere Biology: The Positive Effect of Pharmacologically Active Substances on Telomere Length.

Telomeres are part of chromatin structures containing repeated DNA sequences, which function as protective caps at the ends of chromosomes and prevent DNA degradation and recombination, thus ensuring the integrity of the genome. While telomere length (TL) can be genetically inherited, TL shortening has been associated with ageing and multiple xenobiotics and bioactive substances. TL has been characterised as a reliable biomarker for the predisposition to developing chronic pathologies and their progression. This narrative review aims to provide arguments in favour of including TL measurements in a complex prognostic and diagnostic panel of chronic pathologies and the importance of assessing the effect of different pharmacologically active molecules on the biology of telomeres. Medicines used in the management of cardiovascular diseases, diabetes, schizophrenia, hormone replacement therapy at menopause, danazol, melatonin, and probiotics have been studied for their positive protective effects against TL shortening. All these classes of drugs are analysed in the present review, with a particular focus on the molecular mechanisms involved.

Adult telomere length is positively correlated with survival and lifetime reproductive success in a wild passerine.

Explaining variation in individual fitness is a key goal in evolutionary biology. Recently, telomeres, repeating DNA sequences capping chromosome ends, have gained attention as a biomarker for body state, physiological costs, and senescence. Existing research has provided mixed evidence for whether telomere length correlates with fitness, including survival and reproductive output. Moreover, few studies have examined how the rate of change in telomere length correlates with fitness in wild populations. Here, we intensively monitored an insular population of house sparrows, and collected longitudinal telomere and life history data (16 years, 1225 individuals). We tested whether telomere length and its rate of change predict fitness measures, namely survival, lifespan and annual and lifetime reproductive effort and success. Telomere length positively predicted short-term survival, independent of age, but did not predict lifespan, suggesting either a diminishing telomere length-survival correlation with age or other extrinsic factors of mortality. The positive association of telomere length with survival translated into reproductive benefits, as birds with longer telomeres produced more genetic recruits, hatchlings and reared more fledglings over their lifetime. In contrast, there was no association between telomere dynamics and annual reproductive output, suggesting telomere dynamics might not reflect the costs of reproduction in this population, potentially masked by variation in individual quality. The rate of change of telomere length did not correlate with neither lifespan nor lifetime reproductive success. Our results provide further evidence that telomere length correlates with fitness, and contribute to our understanding of the selection on, and evolution of, telomere dynamics.

Potential to use of viral reverse transcriptase inhibitors in oncology

In preparing the review, articles on the functioning of the reverse transcriptase enzyme of endogenous repeat sequences LINE1, the mechanisms of action and antitumor activity of viral reverse transcriptase inhibitors. Articles available in the biomedical literature information databases SciVerse Scopus, PubMed, Web of Science, Russian Science CitationIndex (RSCI) were analyzed. The review used information from 140 publications, of which 95 and 39 were published, respectively, over the last ten and three years, 2 articles present the results of clinical studies, and 45 articles refer to results demonstrating the anticancer properties of the studied compounds in various models in vitro and in vivo. Aim. Based on data on the functional properties of the reverse transcriptase enzyme of endogenous repeat sequences LINE1 (long interspersed nuclear elements 1), analyze the potential use of viral reverse transcriptase inhibitors in oncology, presenting their classification and main mechanisms of action. About 98 % of the human genome consists of repetitive sequences, most of which are represented by mobil genetic elements, the activation of which leads to increased genome instability. These include long (LINE) and short (SINE) interspersed nuclear element repeated DNA sequences interspersed nuclear elements, respectively, which occupy about 45 % of the human genome. Increased expression levels of these sequences in the genome have been identified in many forms of malignant neoplasms. Their transposition occurs due to the expression of LINE1-encoded reverse transcriptase, whichis homologous to viral reverse transcriptase. To date, reverse transcriptase inhibitors of viruses of nucleoside and non-nucleoside structure have been developed and are successfully used in the clinic. These drugs demonstrate an inhibitory effect on both LINE1 reverse transcriptase and telomerase, which provides the tumor cell with the ability to overcome replicative senescence. Due to these properties, these compounds are expected to exhibit both their own antitumor activity and increase the sensitivity of tumor cells to the therapy of malignant neoplasms, which is experimentally confirmed in models of malignant tumors in vitro and in vivo. Use of reverse transcriptase inhibitors in combination therapy seems advisable both to prevent further genome rearrangements caused by LINE1 and to suppress the survival of tumor cells by inhibiting telomerase activity.

Investigating the potential of X chromosome shredding for mouse genetic biocontrol

CRISPR-Cas9 technology has facilitated development of strategies that can potentially provide more humane and effective methods to control invasive vertebrate species, such as mice. One promising strategy is X chromosome shredding which aims to bias offspring towards males, resulting in a gradual and unsustainable decline of females. This method has been explored in insects with encouraging results. Here, we investigated this strategy in Mus musculus by targeting repeat DNA sequences on the X chromosome with the aim of inducing sufficient DNA damage to specifically eliminate X chromosome-bearing sperm during gametogenesis. We tested three different guide RNAs (gRNAs) targeting different repeats on the X chromosome, together with three male germline-specific promoters for inducing Cas9 expression at different stages of spermatogenesis. A modest bias towards mature Y-bearing sperm was detected in some transgenic males, although this did not translate into significant male-biasing of offspring. Instead, cleavage of the X chromosome during meiosis typically resulted in a spermatogenic block, manifest as small testes volume, empty tubules, low sperm concentration, and sub/infertility. Our study highlights the importance of controlling the timing of CRISPR-Cas9 activity during mammalian spermatogenesis and the sensitivity of spermatocytes to X chromosome disruption.

Breaking down causes, consequences, and mediating effects of telomere length variation on human health

BackgroundTelomeres form repeated DNA sequences at the ends of chromosomes, which shorten with each cell division. Yet, factors modulating telomere attrition and the health consequences thereof are not fully understood. To address this, we leveraged data from 326,363 unrelated UK Biobank participants of European ancestry.ResultsUsing linear regression and bidirectional univariable and multivariable Mendelian randomization (MR), we elucidate the relationships between leukocyte telomere length (LTL) and 142 complex traits, including diseases, biomarkers, and lifestyle factors. We confirm that telomeres shorten with age and show a stronger decline in males than in females, with these factors contributing to the majority of the 5.4% of LTL variance explained by the phenome. MR reveals 23 traits modulating LTL. Smoking cessation and high educational attainment associate with longer LTL, while weekly alcohol intake, body mass index, urate levels, and female reproductive events, such as childbirth, associate with shorter LTL. We also identify 24 traits affected by LTL, with risk for cardiovascular, pulmonary, and some autoimmune diseases being increased by short LTL, while longer LTL increased risk for other autoimmune conditions and cancers. Through multivariable MR, we show that LTL may partially mediate the impact of educational attainment, body mass index, and female age at childbirth on proxied lifespan.ConclusionsOur study sheds light on the modulators, consequences, and the mediatory role of telomeres, portraying an intricate relationship between LTL, diseases, lifestyle, and socio-economic factors.

Late-life aerobic exercise reverses DNA damage and telomere dysfunction in non-atheroprone aortic regions with advanced age

Cellular senescence, a state of cell cycle arrest, contributes to arterial aging. The dominant factor driving cellular senescence is DNA damage. Moreover, telomeres, a repeated DNA sequence at the end of chromosomes, are especially vulnerable to DNA damage induced by DNA-damaging stimuli such as atheroprone shear stress. Although the adoption of aerobic exercise in later life is an effective strategy to mitigate arterial aging, its direct relationship with DNA damage and telomere dysfunction has yet to be elucidated. Additionally, the impact of late-life aerobic exercise, particularly in regions exposed to atheroprone and non-atheroprone shear stress, may not yield uniform benefits, requiring further exploration. We sought to investigate the effects of late-life aerobic exercise on DNA damage and telomere dysfunction in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in aortic regions exposed to distinct shear stress patterns such as the major aortic arch, minor aortic arch, and thoracic aorta. Fifteen male C57BL6 (22-23 mo) were given access to voluntary running wheel (VWR) for 4 weeks. They were classified as high-running (HR), moderate-running (MR), and low-running (LR) groups based on their habitual VWR distances (n=5 per group). Habitual VWR distance was greater in HR and MR compared to LR (75.9±11.2 and 21.3±1.6 vs. 5.8±2 m/day; P≤0.0003). Maximal treadmill running distance improved in HR, did not change in MR, and diminished in the LR (HR, MR vs. LR; P≤0.035) and was correlated to habitual VWR distance at 1-month post-intervention (R2=0.252, P=0.056). Immunofluorescence-fluorescent in situ hybridization was performed to detect the abundance of 53BP1 foci, a marker of DNA damage, and colocalization to telomeres, a measure of telomere-specific DNA damage known as telomere dysfunction-induced foci (TIF). The percentage of ECs containing 53BP1 foci and TIF was lower in the non-atheroprone aortic regions, i.e. major aortic arch and thoracic aorta, of HR compared to LR (P≤0.002), but there was no difference in the atheroprone, i.e. minor aortic arch (P≥0.899). A strong to moderate correlation was observed between 53BP1 foci and habitual VWR distance in major aortic arch and thoracic aorta (R2=0.584 and R2=0.54, P≤0.002) but only between VWR and TIF in the thoracic aorta (R2=0.356, P=0.019). The 53BP1 foci and TIF in VSMCs were comparable across groups in all aortic regions (P≥0.102). Likewise, Telomere length in ECs and VSMCs were similar across groups regardless of aortic region (P≥0.296). In conclusion, our findings suggest that increased level of habitual aerobic exercise in later life has a beneficial impact on DNA damage and telomere dysfunction in ECs, particularly in aortic regions exposed to extended periods of non-atheroprone shear stress. Notably, the volume of aerobic exercise was inversely associated with DNA damage and telomere dysfunction. These effects were independent of telomere length. The improvement in DNA damage and telomere dysfunction associated with habitual aerobic exercise were specific to ECs, as VSMCs remained unaffected in all aortic regions and results were independent of telomere length. Funded in part by awards from National Institutes of Health Awards R01 AG060395, R01 AG077751, R01 AG076748, T32 HL007576, T32 HL139451, Veteran's Affairs Merit Review Award I01 BX004492 and Nora Eccles Treadwell Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Strategies for tularemia pathogen survival, spread and virulence

The bacterium Francisella tularensis is the etiological agent of tularemia, a natural focal, especially dangerous infection, which, “thanks to” its low infectious dose and ability to be transmitted to humans via all possible routes, is a potential bioterrorism agent. This pathogen has been known to mankind for over a hundred years, but it is still impossible to prevent massive human disease outbreaks and sporadic incidence cases, whereas tularemia diagnosis may be verified within several days-to-weeks. The basis for tularemia causative agent virulence is based on its ability to disrupt phagocyte function. In animals and humans, the various Francisella tularensis systems work together to bypass host immune system, attach to and enter eukaryotic cells, block phagosome-lysosome fusion, multiply in various host cells without being detected, inhibit their destruction and cause host cell death to release bacteria and infect neighboring tissue cells, thus developing an infectious disease in different organs. This is achieved through a unique complement-dependent penetration process into host cell, called loop phagocytosis, and an unusual inert endotoxin as well as variation in diverse forms of “free” lipid A modifications and lipid A in the LPS composition, its dynamic acyl chain length regulation, and specifically combined regulatory factors to induce the “pathogenicity island” protein synthesis. Accumulated point mutations, intragenomic rearrangements, deletions, insertions, duplications, transpositions, gene degradation, variation in the number of copies in repeated DNA sequences, as well as homologous and non-homologous recombinations underlie a markedly expanded potential for existence of the tularemia causative agent: they contribute to the holarctic subspecies strain survival in varying conditions, including osmotic shock, to form multiple resistance to various toxic substances and alter F. tularensis subspecies virulence. Analyzing a whole body of publications on the abovementioned aspects for tularemia causative agent life activity attempts to combine the differences, structural features and “tricks” of the F. tularensis species cells allowing them to be a powerful pathogen, with a high potential to adapt upon low pathogen variability and a limited genome length compared with other specially dangerous bacteria.

Proximal telomeric decompaction due to telomere shortening drives FOXC1-dependent myocardial senescence.

Telomeres, TTAGGGn DNA repeat sequences located at the ends of eukaryotic chromosomes, play a pivotal role in aging and are targets of DNA damage response. Although we and others have demonstrated presence of short telomeres in genetic cardiomyopathic and heart failure cardiomyocytes, little is known about the role of telomere lengths in cardiomyocyte. Here, we demonstrate that in heart failure patient cardiomyocytes, telomeres are shortened compared to healthy controls. We generated isogenic human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with short telomeres (sTL-CMs) and normal telomeres (nTL-CMs) as model. Compared to nTL-CMs, short telomeres result in cardiac dysfunction and expression of senescent markers. Using Hi-C and RNASeq, we observe that short telomeres induced TAD insulation decrease near telomeric ends and this correlated with a transcription upregulation in sTL-CMs. FOXC1, a key transcription factor involved in early cardiogenesis, was upregulated in sTL-CMs and its protein levels were negatively correlated with telomere lengths in heart failure patients. Overexpression of FOXC1 induced hiPSC-CM aging, mitochondrial and contractile dysfunction; knockdown of FOXC1 rescued these phenotypes. Overall, the work presented demonstrate that increased chromatin accessibility due to telomere shortening resulted in the induction of FOXC1-dependent expression network responsible for contractile dysfunction and myocardial senescence.

Cdc13 exhibits dynamic DNA strand exchange in the presence of telomeric DNA.

Telomerase is the enzyme that lengthens telomeres and is tightly regulated by a variety of means to maintain genome integrity. Several DNA helicases function at telomeres, and we previously found that the Saccharomyces cerevisiae helicases Hrq1 and Pif1 directly regulate telomerase. To extend these findings, we are investigating the interplay between helicases, single-stranded DNA (ssDNA) binding proteins (ssBPs), and telomerase. The yeast ssBPs Cdc13 and RPA differentially affect Hrq1 and Pif1 helicase activity, and experiments to measure helicase disruption of Cdc13/ssDNA complexes instead revealed that Cdc13 can exchange between substrates. Although other ssBPs display dynamic binding, this was unexpected with Cdc13 due to the reported in vitro stability of the Cdc13/telomeric ssDNA complex. We found that the DNA exchange by Cdc13 occurs rapidly at physiological temperatures, requires telomeric repeat sequence DNA, and is affected by ssDNA length. Cdc13 truncations revealed that the low-affinity binding site (OB1), which is distal from the high-affinity binding site (OB3), is required for this intermolecular dynamic DNA exchange (DDE). We hypothesize that DDE by Cdc13 is the basis for how Cdc13 'moves' at telomeres to alternate between modes where it regulates telomerase activity and assists in telomere replication.

STRIDE-DB: a comprehensive database for exploration of instability and phenotypic relevance of short tandem repeats in the human genome.

Short Tandem Repeats (STRs) are genetic markers made up of repeating DNA sequences. The variations of the STRs are widely studied in forensic analysis, population studies and genetic testing for a variety of neuromuscular disorders. Understanding polymorphic STR variation and its cause is crucial for deciphering genetic information and finding links to various disorders. In this paper, we present STRIDE-DB, a novel and unique platform to explore STR Instability and its Phenotypic Relevance, and a comprehensive database of STRs in the human genome. We utilized RepeatMasker to identify all the STRs in the human genome (hg19) and combined it with frequency data from the 1000 Genomes Project. STRIDE-DB, a user-friendly resource, plays a pivotal role in investigating the relationship between STR variation, instability and phenotype. By harnessing data from genome-wide association studies (GWAS), ClinVar database, Alu loci, Haploblocks in genome and Conservation of the STRs, it serves as an important tool for researchers exploring the variability of STRs in the human genome and its direct impact on phenotypes. STRIDE-DB has its broad applicability and significance in various research domains like forensic sciences and other repeat expansion disorders. Database URL: https://stridedb.igib.res.in.

Targeted Sequencing of Human Satellite 2 Repeat Sequences in Plasma cfDNA Reveals Potential Breast Cancer Biomarkers.

Liquid biopsies are revolutionizing the detection and management of malignant diseases. While repetitive DNA sequences, such as LINE-1 and ALU are established in cell-free DNA (cfDNA) research, their clinical applications remain limited. In this study, we explore human satellite 2 (HSATII), a prevalent repeat DNA sequence in plasma that exhibits increased levels in cancer patients, thereby positioning it as a potential pan-cancer biomarker. We employed targeted sequencing and copy number variation (CNV) analysis using two primer pairs to assess the differential abundance of HSATII sequences in the plasma of breast cancer patients compared to healthy individuals. PCR amplicons of HSATII from 10 patients and 10 control subjects were sequenced, generating 151 bp paired-end reads. By constructing a pooled reference dataset, HSATII copy ratios were estimated in the patients. Our analysis revealed several significant CNVs in HSATII, with certain sequences displaying notable gains and losses across all breast cancer patients, suggesting their potential as biomarkers. However, we observed pronounced fragmentation of cfDNA in cancer, leading to the loss of longer PCR amplicons (>180 bp). While not all observed losses can be attributed to fragmentation artifacts, this phenomenon does introduce complexity in interpreting CNV data. Notably, this research marks the first instance of targeted HSATII sequencing in a liquid biopsy context. Our findings lay the groundwork for developing sequencing-based assays to detect differentially represented HSATII sequences, potentially advancing the field of minimally-invasive cancer screening.

Origin, evolution, and maintenance of gene-strand bias in bacteria.

Gene-strand bias is a characteristic feature of bacterial genome organization wherein genes are preferentially encoded on the leading strand of replication, promoting co-orientation of replication and transcription. This co-orientation bias has evolved to protect gene essentiality, expression, and genomic stability from the harmful effects of head-on replication-transcription collisions. However, the origin, variation, and maintenance of gene-strand bias remain elusive. Here, we reveal that the frequency of inversions that alter gene orientation exhibits large variation across bacterial populations and negatively correlates with gene-strand bias. The density, distance, and distribution of inverted repeats show a similar negative relationship with gene-strand bias explaining the heterogeneity in inversions. Importantly, these observations are broadly evident across the entire bacterial kingdom uncovering inversions and inverted repeats as primary factors underlying the variation in gene-strand bias and its maintenance. The distinct catalytic subunits of replicative DNA polymerase have co-evolved with gene-strand bias, suggesting a close link between replication and the origin of gene-strand bias. Congruently, inversion frequencies and inverted repeats vary among bacteria with different DNA polymerases. In summary, we propose that the nature of replication determines the fitness cost of replication-transcription collisions, establishing a selection gradient on gene-strand bias by fine-tuning DNA sequence repeats and, thereby, gene inversions.

Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome

BackgroundHybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors.ResultsWe used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation “blacklist” in the BED region to reduce errors in downstream analyses.ConclusionsWe first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

Technical Note: A simple FTA® based method for the direct STR amplification of human foetal tissues

Short tandem repeats (STRs) or microsatellites are short, tandemly repeated DNA sequences that involve a repetitive unit of 1–6 bp. DNA isolation and purification from a large number and often compromised samples gives problems to forensic labs for STR typing. Many of the conventional methods used in the isolation and purification of DNA from forensic samples are time consuming, expensive, hazardous for health and are often associated with greater risks of cross contamination. FTA® technology is a method designed to simplify the collection, shipment, archiving and purification of nucleic acid from a wide variety of biological samples. We report a new method for the direct STR amplification which can amplify STR loci from human foetal tissues spotted on FTA cards, bye-passing the need of DNA purification. The STR loci amplified by this method was compared with conventional method of STR profiling and was found absolutely matching. Therefore, this new method is demonstrated to be very useful for fast, less expensive and non- hazardous forensic DNA analysis.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica

Yarrowia lipolytica is widely used in biotechnology to produce recombinant proteins, food ingredients and diverse natural products. However, unstable expression of plasmids, difficult and time-consuming integration of single and low-copy-number plasmids hampers the construction of efficient production pathways and application to industrial production. Here, by exploiting sequence diversity in the long terminal repeats (LTRs) of retrotransposons and ribosomal DNA (rDNA) sequences, a set of vectors and methods that can recycle multiple and high-copy-number plasmids was developed that can achieve stable integration of long-pathway genes in Y. lipolytica. By combining these sequences, amino acids and antibiotic tags with the Cre-LoxP system, a series of multi-copy site integration recyclable vectors were constructed and assessed using the green fluorescent protein (HrGFP) reporter system. Furthermore, by combining the consensus sequence with the vector backbone of a rapidly degrading selective marker and a weak promoter, multiple integrated high-copy-number vectors were obtained and high levels of stable HrGFP expression were achieved. To validate the universality of the tools, simple integration of essential biosynthesis modules was explored, and 7.3 g/L of L-ergothioneine and 8.3 g/L of (2S)-naringenin were achieved in a 5 L fermenter, the highest titres reported to date for Y. lipolytica. These novel multi-copy genome integration strategies provide convenient and effective tools for further metabolic engineering of Y. lipolytica.

A novel detection method based on MIRA-CRISPR/Cas13a-LFD targeting the repeated DNA sequence of Trichomonas vaginalis

BackgroundTrichomonas vaginalis is a protozoan parasite, widely recognized as the most prevalent non-viral sexually transmitted infection (STI) globally. This infection is linked to various complications, including pelvic inflammatory disease, adverse pregnancy outcomes, and an increased risk of acquiring HIV. Current molecular detection methods for T. vaginalis are often costly and technically challenging.MethodsWe developed a novel detection method for T. vaginalis using a multi-enzyme isothermal rapid amplification–clustered regularly interspaced short palindromic repeats (MIRA-CRISPR)/Cas13a-lateral flow device (LFD). This assay targets the repeated DNA sequence (GenBank: L23861.1) of T. vaginalis and is performed at a constant temperature of 37 °C for approximately 1 hour.ResultsThe detection limit of genomic DNA (gDNA) using our protocol was 1 × 10–4 ng/μl. Specificity was confirmed by the absence of cross-reaction with gDNA from various other microorganisms such as Staphylococcus aureus, Lactobacillus taiwanensis, Escherichia coli, Monilia albicans, Giardia lamblia, or Toxoplasma gondii. Among 30 clinical samples tested, the positive rates of T. vaginalis detection were 33.33% (10/30) by wet mount microscopy, 40% (12/30) by nested polymerase chain reaction (PCR), 40% (12/30) by MIRA-CRISPR/Cas13a-LFD, and 40% (12/30) by the culture method. Compared with the culture method, the gold standard for diagnosing trichomoniasis, wet mount microscopy showed a sensitivity of 83.3% and moderate diagnostic agreement (kappa value = 0.87). Both nested PCR and MIRA-CRISPR/Cas13a-LFD exhibited 100% sensitivity and excellent diagnostic agreement (kappa value = 1).ConclusionsThe MIRA-CRISPR/Cas13a-LFD method is a convenient, rapid, stable, and accurate diagnostic tool for detecting T. vaginalis. This method has the potential to enhance the diagnosis and management of vaginitis, offering a significant improvement over existing diagnostic techniques.Graphical