DNA Sequencing Methods Research Articles

CGRclust: Chaos Game Representation for twin contrastive clustering of unlabelled DNA sequences

BackgroundTraditional supervised learning methods applied to DNA sequence taxonomic classification rely on the labor-intensive and time-consuming step of labelling the primary DNA sequences. Additionally, standard DNA classification/clustering methods involve time-intensive multiple sequence alignments, which impacts their applicability to large genomic datasets or distantly related organisms. These limitations indicate a need for robust, efficient, and scalable unsupervised DNA sequence clustering methods that do not depend on sequence labels or alignment.ResultsThis study proposes CGRclust, a novel combination of unsupervised twin contrastive clustering of Chaos Game Representations (CGR) of DNA sequences, with convolutional neural networks (CNNs). To the best of our knowledge, CGRclust is the first method to use unsupervised learning for image classification (herein applied to two-dimensional CGR images) for clustering datasets of DNA sequences. CGRclust overcomes the limitations of traditional sequence classification methods by leveraging unsupervised twin contrastive learning to detect distinctive sequence patterns, without requiring DNA sequence alignment or biological/taxonomic labels. CGRclust accurately clustered twenty-five diverse datasets, with sequence lengths ranging from 664 bp to 100 kbp, including mitochondrial genomes of fish, fungi, and protists, as well as viral whole genome assemblies and synthetic DNA sequences. Compared with three recent clustering methods for DNA sequences (DeLUCS, iDeLUCS, and MeShClust v3.0.), CGRclust is the only method that surpasses 81.70% accuracy across all four taxonomic levels tested for mitochondrial DNA genomes of fish. Moreover, CGRclust also consistently demonstrates superior performance across all the viral genomic datasets. The high clustering accuracy of CGRclust on these twenty-five datasets, which vary significantly in terms of sequence length, number of genomes, number of clusters, and level of taxonomy, demonstrates its robustness, scalability, and versatility.ConclusionCGRclust is a novel, scalable, alignment-free DNA sequence clustering method that uses CGR images of DNA sequences and CNNs for twin contrastive clustering of unlabelled primary DNA sequences, achieving superior or comparable accuracy and performance over current approaches. CGRclust demonstrated enhanced reliability, by consistently achieving over 80% accuracy in more than 90% of the datasets analyzed. In particular, CGRclust performed especially well in clustering viral DNA datasets, where it consistently outperformed all competing methods.

The Role of Bacteria in Pink Stone Discoloration: Insights from Batalha Monastery

The colonization of historical buildings and monuments by fungi, algae, and bacteria is a common phenomenon. This often leads to deterioration processes that cause either visual or structural harm. The Batalha Monastery in Portugal, a UNESCO World Heritage Site, currently shows significant surface changes to the stone architectural elements within both the Founder’s Chapel and the church, including a widespread pink discoloration on the walls and columns. The main goal of this study was to analyze the biological colonization and assess the influence of bacterial communities on the biodeterioration of Ançã limestone, providing valuable insights to help conservators and restorers select the best preservation strategies for the monastery. The prokaryote population was characterized using both high-throughput DNA sequencing and culture-dependent methods and several orange-pink pigment-producing bacteria were identified, for example, Bacillus, Gordonia, Serratia and Methylobacterium, as well as Halalkalicoccus, an abundant archaeal genus. The pink discoloration observed could be due to biofilms created by bacteria that produce pigments, namely carotenoids. Biocolonization tests were performed using stone mock-ups, which were prepared and inoculated with the bacteria isolated in this study. These tests were designed to replicate the natural conditions of the monastery and monitor the colonization process to understand the discoloration phenomenon.

Characterization of Nodulation-Compatible Strains of Native Soil Rhizobia from the Rhizosphere of Soya Bean (Glycine max L.) Fields in South Africa

Prolonged inoculation of soya bean (Glycine max L.) farms with exotic strains of Bradyrhizobium species starting in the 1960s resulted in the establishment of populations of Bradyrhizobium strains in the soils of several soya bean farms in South Africa. With the increasing number of new soya bean genotypes in the country, it is challenging to determine which genotypes are highly compatible with a given rhizobium strain. In this study, we investigated the symbiotic compatibility of native rhizobial isolates and the strains from the South African Rhizobium Culture Collection (SARCC) on ten selected locally available soya bean genotypes. A glasshouse soil trap experiment using soil samples collected from Lothair, Bothaville, and Standerton was performed on five cultivars. The trapped rhizobial strains were further screened in the glasshouse to authenticate their nodulation compatibility with the different soya bean cultivars. The rhizobial strains showed significant nodulation compatibility with the selected cultivars. These strains were also tested for beneficial traits in vitro and characterized using DNA sequencing methods to elucidate their taxonomic identity. Some of the most nodulation-compatible strains characterized as Bradyrhizobium and Sinorhizobium species exhibited significant symbiotic performance in terms of plant biomass, nodule number, and nodule dry weight. The study generated valuable data that provide information on the extent of symbiotic compatibility of some of the existing cultivars used in South Africa with native rhizobia and whether inoculation of soya bean with commercial products is vital on some soya bean farms.

Workplace genetic testing: which employees are likely to participate, what are their concerns with employer sponsorship, and which design features could reduce barriers and increase participation?

Voluntary genetic testing (GT) leverages low-cost DNA sequencing and other testing methods to provide genetic risk screening for healthy individuals. Given the potential to prevent disease and promote health, some employers now offer GT as an employee benefit (workplace GT, or wGT), but participation remains low. To investigate facilitators and barriers to wGT participation, we conducted one of the first representative surveys of working U.S. adults on this topic (n = 958). We assessed factors that could influence participation, including: sponsoring entity (health provider or employer), program design, and individual demographics. Two-thirds (68%) of respondents indicated willingness to participate in some type of GT, but only half (49%) expressed willingness to participate through their employer. Women were 60% more willing to participate than men, and individuals with previous genetic testing experience were 143% more willing to participate than those without such experience. Across all demographic groups, certain GT program design features tended to increase or decrease willingness to participate. The ability to have one's data deleted from the GT database increased willingness most often (true for 67% of respondents), while selling data to pharmaceutical companies decreased willingness most often (true for 63% of respondents).

Emergence of Nigrospora Leaf Blight as Major Biotic Stress in South Eastern Coastal Region of India

Background: Nigrospora is one of the diversified pathogens with wide host range. Endophytic nature of Nigrospora oryzae has been reported previously. Daily variations in the climate led to the emergence of virulent infections from previously expressed weak ones. Nigrospora oryzae infestation comes as a major biotic stress under this present investigation. Methods: A study was conducted to examine the variability of Nigrospora oryzae by collecting isolates from various locations in the south coastal region of Odisha. The level of virulence was assessed and analyzed between 2021 and 2023 to anticipate the emergence and progression of disease. The pathogen was identified using molecular techniques through ITS-r DNA sequencing method. Result: Disease incidence was observed as a critical blast-like appearance, but greater in size, distinguishing it from blast. In the experiment, incomplete filling of grains was highest in MTU-7029 (39.73 plant-1) followed by Niranjana and Gobindobhog; disease incidence was recorded to be highest in Niranjana (60.03%) followed by MTU-7029 and Barsha; and grain yield plant-1 was observed to be highly affected in Gobindobhog (14.58 g plant-1 followed by MTU-7029 and Niranjana. Molecular confirmation was established by ITS-rDNA sequencing, resulted sequence NO 9 (528 bp)- OP592217 and NO 15 (519 bp)- OP592218, exactly matches gene bank accession numbers- MH748173 (India) and ON514037 (India) of Nigrospora database, with 99% accuracy.

Visualization Methods for DNA Sequences: A Review and Prospects.

The efficient analysis and interpretation of biological sequence data remain major challenges in bioinformatics. Graphical representation, as an emerging and effective visualization technique, offers a more intuitive method for analyzing DNA sequences. However, many visualization approaches are dispersed across research databases, requiring urgent organization, integration, and analysis. Additionally, no single visualization method excels in all aspects. To advance these methods, knowledge graphs and advanced machine learning techniques have become key areas of exploration. This paper reviews the current 2D and 3D DNA sequence visualization methods and proposes a new research direction focused on constructing knowledge graphs for biological sequence visualization, explaining the relevant theories, techniques, and models involved. Additionally, we summarize machine learning techniques applicable to sequence visualization, such as graph embedding methods and the use of convolutional neural networks (CNNs) for processing graphical representations. These machine learning techniques and knowledge graphs aim to provide valuable insights into computational biology, bioinformatics, genomic computing, and evolutionary analysis. The study serves as an important reference for improving intelligent search systems, enriching knowledge bases, and enhancing query systems related to biological sequence visualization, offering a comprehensive framework for future research.

Machine Learning Approaches for Microorganism Identification, Virulence Assessment, and Antimicrobial Susceptibility Evaluation Using DNA Sequencing Methods: A Systematic Review.

Microbial infections pose a substantial global health challenge, particularly impacting immunocompromised individuals and exacerbating the issue of antimicrobial resistance (AMR). High virulence of pathogens can lead to severe infections and prolonged antimicrobial treatment, increasing the risk of developing resistant strains. Integrating machine-learning (ML) with DNA sequencing technologies offers potential solutions by enhancing microbial identification, virulence assessment, and antimicrobial susceptibility evaluation. This review explores recent advancements in these integrated approaches, addressing current limitations and identifying gaps in the literature. A comprehensive literature search was conducted across databases including PubMed, Scopus, Web of Science, and IEEE Xplore, covering publications from January 2014 to June 2024. Using a detailed Boolean search string, relevant studies focusing on ML applications in microorganism identification, antimicrobial susceptibility testing, and microbial virulence were included. The screening process involved a two-stage review of titles, abstracts, and full texts, with data extraction and critical appraisal performed using the QIAO tool. Data were analyzed through narrative synthesis to identify common themes and innovations. Out of 1,650 initially identified records, 19 studies met the inclusion criteria. These studies primarily focused on AMR, with additional research on microbial virulence and identification. Machine learning algorithms such as Random Forest, Support Vector Machines, and Convolutional Neural Networks, combined with DNA sequencing techniques like Whole Genome Sequencing and Metagenomic Sequencing, demonstrated significant advancements in predictive accuracy and efficiency. High-quality studies achieved impressive performance metrics, including F1-scores up to 0.88 and AUC scores up to 0.96. The integration of ML and DNA sequencing technologies has significantly enhanced microbial analysis, improving the identification of pathogens, assessment of virulence, and evaluation of antimicrobial susceptibility. Despite advancements, challenges such as data quality, high costs, and model interpretability persist. This review highlights the need for continued innovation and provides recommendations for future research to address these limitations and improve disease management and public health strategies. The systematic review is registered with PROSPERO (CRD42024571347).

Leucine repeat polymorphism in CNDP1 gene (D18S880) and Type 2 Diabetic kidney disease in South Indians

The global concern is the alarming increase in the high proportion of Diabetes Mellitus affecting nearly all populations which in turn emphasized the urge to investigate the role of susceptibility genes contributing to the disease burden. The highly vulnerable genes accountable for promoting type 2 Diabetic Kidney Disease (T2DKD) in South Indians are not well studied. This study is an attempt to research the possible association between (CTG)n trinucleotide repeats in Carnosinase or Carnosine dipeptidase 1 (CNDP1) gene and Diabetic Kidney disease (DKD) in South Indian population. The repetitive sequence of CTG in CNDP1 gene was amplified from the genomic DNA of total 285 [T2DKD, n:85; Type 2 Diabetes Mellitus (T2DM), n:100; healthy controls (HC), n:100] subjects using DNA sequencing method and was examined by a case-control approach. The significant trinucleotide repeats in our study were homozygous 5/5 leucine, heterozygous 5/6 leucine and homozygous 6/6 leucine. T2DKD had a lower frequency of homozygous 5/5 leucine genotype (23.5%) than T2DM (37%, P=0.047) and HC (41%, P=0.011). Likewise, five leucine allele (5L) recurrence was lower in T2DKD (45.9%) when compared with T2DM (59.5%, P=0.008) and HC (62.5%, P=0.001). T2DKD showed lower frequencies of homozygous 5/5 leucine genotype and five leucine allele (5L) of CNDP1 gene when compared to T2DM and HC. These findings established that this allele may offer defence against the onset of kidney disease in South Indians.

Vaginal Microbiome and the Risk of Preterm Birth in Women Living With HIV: A Scoping Review.

There are sparse data on the role of thevaginal microbiome (VMB) in pregnancy among pregnant women living with HIV (PWLWH) and its association with spontaneous preterm birth (sPTB). We conducted a scoping review to assess associations between vaginal microbiota and sPTB among PWLWH. Three studies were included, representing a total of 180 PWLWH out of 652 total pregnancies. All studies used modern DNA sequencing methods (16S rRNA amplification, metagenomics, or metatranscriptomics). PWLWH had higher VMB richness and diversity compared to HIV-uninfected pregnant women and higher sPTB rates in two of three studies. A higher proportion of sPTB among PWLWH was observed in those with Lactobacillus-deficient, anaerobe-dominant vaginal microbiota. In two of three studies, higher concentrations of vaginal inflammation markers were associated with increased VMB richness and diversity. HIV status was independently associated with sPTB. It is unclear if increased vaginal microbial diversity among PWLWH or increased vaginal inflammation contributes more to PTB, but HIV does appear to alter the VMB in pregnant individuals and may also affect PTB rates in microbiome-independent pathways. Given the limited number of studies, heterogeneity in sample size, sample collection methods, and inconsistent results it is difficult to causally link HIV, VMB, inflammatory cytokines, and sPTB.

Mitochondrial genetic variations in leukemia: a comprehensive overview.

Leukemias are a group of heterogeneous hematological malignancies driven by diverse genetic variations, and the advent of genomic sequencing technologies facilitates the investigation of genetic abnormalities in leukemia. However, these sequencing-based studies mainly focus on nuclear DNAs. Increasing evidence indicates that mitochondrial dysfunction is an important mechanism of leukemia pathogenesis, which is closely related to the mitochondrial genome variations. Here, we provide an overview of current research progress concerning mitochondrial genetic variations in leukemia, encompassing gene mutations and copy number variations. We also summarize currently accessible mitochondrial DNA (mtDNA) sequencing methods. Notably, somatic mtDNA mutations may serve as natural genetic barcodes for lineage tracing and longitudinal assessment of clonal dynamics. Collectively, these findings enhance our understanding of leukemia pathogenesis and foster the identification of novel therapeutic targets and interventions.

DNA methylation in recovery of Maize (Zea Mays L.) from maize streak

DNA methylation influences regulation of gene expression during cell development and tissue differentiation in plants. This study therefore discussed the role of DNA methylation in the recovery of maize from Maize Streak Diseases (MSD). Bisulfite treatment and DNA sequencing methods were carried out on the 2nd, 4th 6th and 8th white to yellowish streaking leaves of five infected maize varieties (DMR-ESR-Y, TZEBR, and ART/98/SW6) to assess the disease severity using digital phenotyping. The bisulfite treatment utilizes various sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulfite under acidic conditions in which cytosine is converted to uracil, whereas 5-MeC remains unreactive. This study showed that DMR-ESR-Y variety significantly (p<0.05) tolerated best for MSD, while TZEBR and ART/98/SW6 varieties slightly tolerated MSD but were not significantly different. The analysis of the images from digital phenotyping revealed that DMR-ESR-Y variety had the highest level of resistance, followed by TZEBR while ART/98/SW6 showed mild resistance and DMR-LSR-Y had the least. The leaf length had strong positive correlation with leaf width (r=0.94), number of leaves(r=0.64), plant height (r=0.96) and stem height(r=0.96). The methylation patterns in bisulfite sequences across the same leaf positions were significantly different (p<0.05), thus suggesting that CG methylation could contribute to transcriptional gene silencing plant recovery mechanism in maize.

Exploring the Antimicrobial Resistance Profile of Salmonella typhi and Its Clinical Burden.

Background: Typhoid fever caused by Salmonella enterica serovar Typhi (S. typhi) continues to pose a significant risk to public health in developing countries, including Pakistan. This study investigated the epidemiological factors linked to suspected and confirmed S. typhi infections in Peshawar's hospital population. Methodology: A total of 5735 blood samples of patients with suspected enteric fever were collected from September 2022 to November 2023. S. typhi infection was confirmed using microbiological culture of blood samples, biochemical-based tests, and DNA-sequencing methods. Drug sensitivity testing on cultures was conducted as per the CLSI guidelines. Chi-square tests were used to analyze the clinical and epidemiologic characteristics of 5735 samples stratified by S. typhi infection status, and risk factors were assessed by applying logistic regression models to estimate odds ratios (ORs). Results: The number of confirmed typhoid fever cases in this hospital-based study population was 691 (/5735, 12.0%), more prevalent in males (447/3235 13.8%) and children (0-11 years) (429/2747, 15.6%). Compared to children, the risk of S. typhi infection was lower in adolescence (adjusted OR = 0.52; 95% CI: 0.42-0.66), adulthood (19-59 years; aOR = 0.30; 95% CI: 0.25-0.38), and older adulthood (aOR = 0.08; 95% CI: 0.04-0.18) (p < 0.001). Compared to males, the risk of S. typhi infection was lower in females (aOR = 0.67; 95% CI = 0.56-0.80; p = 0.002). Living in a rural residence (compared to urban) was associated with a higher risk of infection (aOR = 1.38; 95% CI: 1.16-1.63; p = 0.001), while access to a groundwater source (compared to municipal water supply) led to a lower risk (aOR = 0.56; 95% CI: 0.43-0.73; p = 0.002). Vaccination demonstrated a robust protective effect (aOR = 0.069; 95% CI = 0.04-0.11, p = 0.002). For those with typhoid infections, clinical biomarker analysis revealed the presence of leucopenia (65/691, 9.4%), thrombocytopenia (130/691, 18.8%), and elevated alanine aminotransferase (ALT) (402/691, 58.2%) and C-reactive protein (CRP) (690/691, 99.9%) levels. Worryingly, among the positive S. typhi isolates, there was a high prevalence of drug resistance (653/691), including multidrug-resistant (MDR 82/691, 11.9%) and extensively drug-resistant types (XDR, 571/691, 82.6%). Conclusions: This study highlights the importance of age, sex, locality, water source, and vaccination status in shaping the epidemiological landscape of S. typhi in the Peshawar district. It implies that expanding vaccination coverage to the broader population of Khyber Pakhtunkhwa province, particularly in the district of Peshawar, would be beneficial.

Translocations, rising populations, and phylogeographic consequences: genomic implications for conservation of introduced Aoudad (Ammotragus lervia) in the southwestern United States

Abstract Over the last century, Texas has been inundated with exotic ungulates, with as many as 67 species represented by captive and free-ranging populations. One of the most successful species to have established free-ranging populations is the Aoudad (Ammotragus lervia). Herein, we expand on previous genetic studies in Aoudad by including new genomic profiles based on double-digest restriction site-associated DNA sequencing methods with the intent of better understanding the effects of &amp;gt;70 years of human-mediated translocations, population expansion, and gene flow among populations. Using a range of 4,338 to 5,529 single-nucleotide polymorphisms obtained from 73 individuals that potentially represented A. l. blainei, A. l. lervia, and A. l. sahariensis, we measured genetic patterns of diversity and connectivity between source populations from California and New Mexico and resultant aoudad populations in Texas. Principal component analyses identified phylogeographically structured populations across Texas. Further, we identified a signature of isolation by distance among Texas populations. To assess geographic regions that facilitated or blocked gene flow between populations, we used estimation of effective migration surfaces, which indicated 3 barriers to movement between populations as well as connectivity among populations in close proximity. Comparison of mtDNA haplogroups and nuclear genotypes among the 3 examined subspecies were discordant, indicating that the current subspecific taxonomic classification needs refinement. Given the vulnerable status of Aoudad in their native range of northern Africa and the exponential population growth and potential competition with native ungulates in Texas, we propose that efforts to manage non-native Aoudad should strive to maintain and preserve unique genetic units while minimizing competition and other detriments to native species in Texas. Conservation efforts are especially important given the vulnerable status of Aoudad within their native range of northern Africa.

Single Nucleotide Polymorphisms in APE1, hOGG1, RAD51 Genes and their Association with Radiotherapy Induced Toxicity among Head and Neck Cancer Patients.

Radiotherapy (RT) is a crucial treatment for head and neck cancer however, it causes adverse reactions to the normal tissue and organs adjacent to target tumor. The present study was carried out to investigate possible association of single nucleotide polymorphism in DNA repair genes with toxicity effects of radiotherapy on normal tissue. Three hundred and fifty head and neck cancer patients receiving radiotherapy treatment were enrolled in this study. The adverse after effects of radiotherapy on the normal tissue in the form of skin reactions were recorded. Single nucleotide polymorphisms of APE1 (rs1130409), hOGG1 (rs1052133) and Rad51 (rs1801320, rs1801321) genes were studied by polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) and direct DNA sequencing methods and their association with development of severe radio-toxicity effects was evaluated logistic regression analysis. The 172G/T polymorphism of Rad51 was 2.85 times higher and significantly associated with skin reactions (OR=2.85, 95% CI: 1.50-5.41; p=0.001) and severe oral mucositis (OR=4.96, 95% CI: 2.40-10.25; p<0.0001). These results suggested that the polymorphic nature of Rad51 is responsible for risk of radiotherapy adverse effects in HNC patients. The variant 326Cys and heterozygous 326Ser/Cys genotype of hOGG1 was significantly associated with high tumor grade (OR=3.16 95% CI: 1.66-5.99; p=0.0004, and OR=3.97 95% CI: 2.15-7.34; p=<0.0001 respectively). The homozygous variant 172TT genotype of Rad51 showed positive association with poor response of both tumor and nodes towards radiotherapy treatment (p=0.007 and p=0.022). Interpretation of our results revealed significant association of rs1801321 SNP of Rad51 with development of adverse toxicity reactions in normal tissue of head and neck cancer patients treated with radiotherapy.

P-029 Genome-wide microRNA expression profiling in human spermatozoa and its relation to sperm quality

Abstract Study question Does the microRNA (miRNA) expression profile in human spermatozoa change with their morphological appearance? Summary answer This study revealed miRNA expression associated with good-quality sperm classified based on their morphology. These miRNAs could represent efficient biomarkers to evaluate male fertility status. What is known already We previously published a scoring scale where the spermatozoa with a Score 6 (Good spermatozoon) display normal head shape with symmetrical nuclear no extrusion and/or no invagination of the nuclear membrane, without any vacuole and normal base. The spermatozoa with a Score 0, display a nuclear-shape disorder with an abnormal base and at least one large vacuole (Bad spermatozoon). Despite the great magnitude of studies investigating the miRNA role in spermatogenesis, limited work has been done to evaluate it as a potential biomarker for sperm quality in relation to their morphology. Study design, size, duration In a prospective study from March 2023 to May 2023, twenty semen samples were collected. The samples were categorized according to their scoring morphology into Score 6 and Score 0. Ten samples (five with a Score 6 and five with a Score 0) were used for the analysis by microarray and ten (five with a Score 6, and five with a Score 0) for the miRNA expression analysis by RT-qPCR. Participants/materials, setting, methods Semen samples were scored according to their high-magnification morphology and stored at -80 °C. We used miRNA microarray chip to analyze miRNA profile. Statistical significance was evaluated by Significance Analysis of Microarrays, screened by Fold change&amp;gt;2 and p-values ≤ 0.05. The correlation between miRNAs and their corresponding mRNA targets was analyzed using in silico prediction algorithms. The methylome was studied using the DNA sequencing method. TaqMan miRNA assays were used to confirm the miRNA results. Main results and the role of chance We perform a robust transcriptomic analysis to define unique miRNA signatures able to identify the spermatozoa that have high scores regardless of morphological appearance at high magnification. There were 86 significantly overexpressed in Score 6 and 21 miRNAs in Score 0. Score 6-spermatozoa had significantly higher levels of hsa-miR-132 (FCx41, p=1.68E-8), hsa-miR-1973 (FCx32, p=6.19E-8), hsa-miR-34 (FCx30, p=8.43E-8), hsa-miR-25 (FCx27, p=1.30E-6), hsa-miR-342 (FCx24, p=1.57E-5), hsa-miR-15 (FCx14, p=8.56E-6), hsa-miR-125 (FCx13, p=6E-4), hsa-miR-30 (FCx12, p = 3.75E-6), than Score 0. The miRNAs overexpressed in Score 6 are predicted to target 910 messenger RNA involved in biological functions of the spermatozoa and several signaling pathways, such as mTOR, p53, and MAPK signaling pathways, as well as cell Intrinsic pathway for apoptosis, cellular senescence, and oxidative stress. Whereas genes targeted by score 0-miRNAs were implicated in the regulation of cellular growth and proliferation. Our data indicate a difference in methylation of the promoter region of miRNAs, and their validated target genes were observed between score 6 and score 0. Aberrant expression of the Score 6-miRs or their targets could be due to the methylation status of the promoter region and indicative of a defect in spermatogenesis. Limitations, reasons for caution This work provides a foundation for the interpretation of miRNA changes associated with spermatozoa morphological appearance. The data must be validated by TaqMan miRNA assays in a higher number of semen samples. Moreover, the molecular network of miRNA-targeted gene pathways also merits investigation. These experiments are currently ongoing. Wider implications of the findings The profiling of miRNAs repertoire in scored spermatozoa morphology opens new perspectives to explore male fertility status and provides a biomarker panel for sperm analysis during the ART procedure. Larger well-controlled investigations employing microRNAome approaches are needed to validate these conclusions. Trial registration number not applicable

Investigating the relationship between postoperative radiotherapy and intestinal flora in rectal cancer patients: a study on efficacy and radiation enteritis.

This study aimed to investigate the impact of radiation therapy and radiation enteritis on intestinal flora, providing insights for treatment and prevention. Fecal samples were collected from 16 patients undergoing pelvic radiotherapy at Qingdao Hiser Hospital Affiliated of Qingdao University (Qingdao Traditional Chinese Medicine Hospital). Samples were collected before and after radiotherapy (27-30Gy), and analyzed using DNA sequencing and biostatistical methods. Patients with radiation enteritis showed increased α-diversity and β-diversity of intestinal flora compared to those without radiation enteritis. Differences in flora composition were observed, with higher abundance of secondary pathways such as amino acid metabolism, carbohydrate metabolism, cofactors and vitamins metabolism, and lipid metabolism. The study revealed that patients developing radiation enteritis during pelvic radiation therapy had increased diversity and abundance of intestinal flora compared to those who did not develop radiation enteritis. Additionally, patients without radiation enteritis showed significantly higher diversity and abundance of intestinal flora post-radiation compared to pre-radiation.

HLA Association among Thai Patients with Diffuse and Limited Cutaneous Systemic Sclerosis.

This study aimed to clarify the association of HLA Class I and II with dcSSc and lcSSc in Thais. HLA typing for 11 gene loci (Class I: HLA-A, B and C, and Class II [HLA-DR, DP and DQ]) was carried out using the Next Generation DNA Sequencing method (three fields) in 92 Thai patients with systemic sclerosis (55 dcSSc, 37 lcSSc) and 135 healthy controls (HCs). The distribution of HLA alleles in patients with dcSSc and lcSSc was compared. When compared with HCs, the AF of A*24:02:01, A*24:07:01, B*27:04:01 and B*27:06 showed an increasing trend in lcSSc patients without statistical significance. DRB1*15:02:01, DRB5*01:02:01, DQA1*01:01:01, DQB1*05:01:24, DPA1*02:01:01 and DPB1*13:01:01 increased significantly in dcSSc patients. DQB1*05:01:24 and DPB1*13:01:01 also increased significantly in lcSSc patients, but less significantly than in dcSSc patients. The association of DPB1*05:01:01 with lcSSc was significantly protective. HLA-A*24:02:01, B*27:06 and C*03:04:01 formed a three-locus haplotype that also constituted an eight-locus haplotype with DRB1*15:02:01, DQA1*01:01:01, DQB1*05:01:24, DPA1*02:01:01 and DPB1*13:01:01. There was a possibility that HLA Class I would play a role in the pathogenesis of lcSSc, while Class II played more of a role in the dcSSc in Thai patients.

From Sanger to genome sequencing - an overview of DNA sequencing technologies

There is no technique that would make a greater contribution to the development of genetics, molecular biology and medicine than DNA sequencing. For many years, the method based on enzymatic DNA synthesis developed by Frederic Sanger was the gold standard in this area. At the end of the 20th century, there was a dynamic development of next-generation sequencing (NGS) technologies, which ended the era of single gene analysis and initiated the era of genome sequencing. Despite fierce competition, one NGS technology has practically completely dominated the global market. In the article, we present our own review of DNA sequencing methods, starting from the Sanger method to high-throughput second- and third-generation sequencing technologies, with particular emphasis on those that have achieved commercial success. We present their short history, principles of operation, technical possibilities, applications and limitations. In the summary, we reveal how much human genome sequencing costs at the current stage of the genomic revolution and outline the prospects for further development of genomics.

Genetic structure of a narrowly distributed species Chimonobambusa tumidissinoda in the Yunnan-Guizhou Plateau, and its implications for conservation

Chimonobambusa tumidissinoda is a narrowly distributed and endangered bamboo species, which is endemic to the Yunnan-Guizhou Plateau and possesses significant ecological and economic value, particularly due to its delectable shoots. In this study, we employed the restriction site-associated DNA sequencing (RAD-seq) method to elucidate the population structure, genetic diversity, and demographic history of C. tumidissinoda across its entire distribution range. Unlike the genetic pattern revealed previously, three distinct genetic groups within the C. tumidissinoda samples were consistently discriminated based on multiple analyses, and low levels of genetic diversity (HO = 0.085, HE = 0.115, π = 0.116) were found. Furthermore, moderate genetic differentiation (FST = 0.070 - 0.095; PhiPT = 0.129 - 0.156) was detected among these groups with Mantel tests suggesting that isolation by distance (IBD) could potentially drive population genetic differentiation in this species. Moreover, demographic analyses incicated that C. tumidissinoda likely experienced a bottleneck event since the Pliocene, which may be attributed to drier and colder winters in Yunnan resulting from intensified Asian monsoon activity. Based on these findings, we suggest that the endangered status of C. tumidissinoda has been underestimated. Conservation measures should be implemented both in situ and ex situ to enhance its adaptability towards future climate fluctuations. Additionally, seed collection for germplasm storage is recommended.

Clinical characteristics and antimicrobial susceptibility profiles of Mycobacterium abscessus and Mycobacterium massiliense pulmonary infection

ObjectivesMycobacterium abscessus complex (MABC) is the most common rapidly growing Mycobacterium species in structural pulmonary diseases and can be life-threatening. This study aimed to assess the clinical characteristics and drug-susceptibility statuses of different M. abscessus (MAB) subspecies in the Zhejiang Province. MethodsDNA sequencing was used to differentiate clinical MABC subspecies isolates. The Clinical and Laboratory Standards Institute guidelines were used to determine in vitro susceptibility of imipenem-relebactam (IMP-REL), omadacycline, and other conventional antibiotics. Patient clinical characteristics were collected and analysed. ResultsIn total, 139 M. abscessus, 39 Mycobacterium massiliense, and 1 Mycobacterium bolletii isolates were collected, accounting for 77.7%, 21.8%, and 0.5% of the MABC isolates, respectively. Patients with M. abscessus pulmonary disease (M.ab-PD) had higher proportions of older adults, tuberculosis history, chronic pulmonary disease, and malignancy than those with M. massiliense pulmonary disease (M.ma-PD). Patients with M.ab-PD had higher rates of bilateral middle- and lower-lobe involvement than patients with M.ma-PD. Both subspecies showed high resistance rates to doxycycline and moxifloxacin, and clarithromycin-induced resistance was more common in M.ab than in M.ma. IMP-REL resulted in a twofold reduction in the minimum inhibitory concentration (MIC) value compared with imipenem alone among MAB; furthermore, the MIC was lower in M.ab than in M.ma. Omadacycline and tigecycline had comparable in vitro susceptibility, and the MIC showed no statistically significant difference between M.ab and M.ma. ConclusionsM.ab is the most prevalent MABC subspecies in the Zhejiang Province. Patients with M.ab-PD have complex underlying diseases and broader lobar lesions. IMP-REL and omadacycline are promising antibiotics for MABC infection treatment.