Current Issue : July-September Volume : 2025 Issue Number : 3 Articles : 5 Articles
Evolutionary changes in populations of microbes, such as microalgae, cannot be traced using conventional metabarcoding loci as they lack intraspecific resolution. Consequently, selection and competition processes among strains of the same species cannot be resolved without elaborate isolation, culturing, and genotyping efforts. Bamboozle, a new bioinformatic tool introduced here, scans the entire genome of a species and identifies allele- rich barcodes that enable direct identification of different genetic strains from a population using amplicon sequencing of a single DNA sample. We demonstrate its usefulness by identifying hypervariable barcoding loci (< 500 bp) from genomic data in two microalgal species, the diploid diatom Skeletonema marinoi and the haploid chlorophyte Chlamydomonas reinhardtii. Across the two genomes, four and twenty- two loci, respectively, were identified that could in silico resolve all analysed genotypes. All of the identified loci are within protein- coding genes with various metabolic functions. Single nucleotide polymorphisms (SNPs) provided the most reliable genetic markers, and among 54 strains of S. marinoi, three 500 bp loci contained, on average, 46 SNPs, 103 strain- specific alleles, and displayed 100% heterozygosity. This high level of heterozygosity was identified as a novel opportunity to improve strain quantification and detect false positive artefacts during denoising of amplicon sequences. Finally, we illustrate how metabarcoding of a single genetic locus can be used to track abundances of S. marinoi strains in an artificial selection experiment. As future genomic datasets become available and DNA sequencing technologies develop, Bamboozle has flexible user settings enabling optimal barcodes to be designed for other species and applications....
The unique features of mitochondrial DNA (mtDNA), including its circular and multicopy nature, the possible coexistence of wild-type and mutant molecules (i.e., heteroplasmy) and the presence of nuclear mitochondrial DNA segments (NUMTs), make the diagnosis of mtDNA diseases particularly challenging. The extensive deployment of next-generation sequencing (NGS) technologies has significantly advanced the diagnosis of mtDNA-related diseases. However, the vast amounts and diverse types of sequencing data complicate the interpretation of these variants. From sequence alignment to variant calling, NGS-based mtDNA sequencing requires specialized bioinformatics tools, adapted for the mitochondrial genome. This study presents the use of new bioinformatics approaches, optimized for short- and long-read sequencing data, to enhance the accuracy of mtDNA analysis in diagnostics. Two recent and emerging free bioinformatics tools, Mitopore and MitoSAlt, were evaluated on patients previously diagnosed with single nucleotide variants or large-scale deletions. Analyses were performed in Linux-based environments and web servers implemented in Python, Perl, Java, and R. The results indicated that each tool demonstrated high sensitivity and specific accuracy in identifying and quantifying various types of pathogenic variants. The study suggests that the integrated and parallel use of these tools offers a significant advantage over traditional methods in interpreting mtDNA genetic variants, reducing the computational demands, and provides an accurate diagnostic solution....
Chromosome Conformation Capture (3 C) methods, including Hi-C (a high-throughput variation of 3 C), detect pairwise interactions between DNA regions, enabling the reconstruction of chromatin architecture in the nucleus. HiChIP is a modification of the Hi-C experiment that includes a chromatin immunoprecipitation (ChIP) step, allowing genome-wide identification of chromatin contacts mediated by a protein of interest. In mammalian cells, cohesin protein complex is one of the major players in the establishment of chromatin loops. We present an improved cohesin HiChIP experimental protocol. Using comprehensive bioinformatic analysis, we show that a dual chromatin fixation method compared to the standard formaldehyde-only method, results in a substantially better signal-to-noise ratio, increased ChIP efficiency and improved detection of chromatin loops and architectural stripes. Additionally, we propose an automated pipeline called nf-HiChIP (https:// github. com/SFGLab/hichip-nf-pipeline) for processing HiChIP samples starting from raw sequencing reads data and ending with a set of significant chromatin interactions (loops), which allows efficient and timely analysis of multiple samples in parallel, without requiring additional ChIPseq experiments. Finally, using advanced approaches for biophysical modelling and stripe calling we generate accurate loop extrusion polymer models for a region of interest and provide a detailed picture of architectural stripes, respectively....
The diagnosis and monitoring of eosinophilic esophagitis (EoE), a common pediatric pathology, typically involves invasive procedures such as an upper endoscopy with biopsies, imposing a significant burden on patients and healthcare systems. We aimed to assess miR-21-5p and miR-223-3p levels in pediatric EoE patients and evaluate their as potential non-invasive biomarkers of disease activity and response to treatments. We enrolled 13 children with EoE and 8 controls. Plasma and esophageal mucosa samples from patients were collected at diagnosis and after 8–10 weeks of therapy and compared with control samples. After microRNA(miRNA) extraction, the levels of miR-21-5p and miR-223-3p and their relevant target genes were analyzed. Bioinformatic analysis was used to identify the predicted target genes and pathways that are potentially relevant for disease pathophysiology. Plasma levels of miR-21-5p and miR-223-3p were significantly higher in EoE patients than in the controls, reflecting their levels in esophageal mucosa. The target genes of these miRNAs are involved in key signaling pathways (MAPK, Ras, and FoxO), relevant for EoE pathophysiology. Among these, STAT3 (Signal Transducer and Activator of Transcription 3) and PTEN (Phosphatase and Tensin Homolog), which are significantly downregulated in patient esophageal mucosa, are implicated in eosinophilic gastroenteropathies and autoimmune diseases. Following therapy (proton pump inhibitors and/or fluticasone propionate), plasma and tissue expression of both miRNAs significantly decreased and were no longer different from the controls. These microRNAs may serve as complementary non-invasive EoE markers and reduce the need for endoscopy/biopsies....
Background. Whole- genome sequencing of bacterial isolates is increasingly becoming routine in clinical microbiology; however, subsequent analysis often needs to be started by a bioinformatician even for comprehensive pipelines. To increase the robustness of our workflow and free up bioinformatician work hours for development and advanced analysis, we aimed to produce a robust, customizable bioinformatic pipeline for bacterial genome assembly and routine analysis results that could be initiated by non- bioinformaticians. Results. We introduce the RSYD- BASIC pipeline for bacterial isolate sequence analysis and provide a demonstration of its functionality with two datasets composed of publicly available sequences, in which comparable results are obtained in most cases. In some instances, the pipeline provided additional information, corresponding to in vitro results where these could be obtained. In routine use at our department, the pipeline has already yielded clinically relevant results, allowing us to type a variety of bacterial pathogens isolated in our clinical laboratory. We also demonstrate how RSYD- BASIC results aided in disproving a potential outbreak. Conclusion. With the RSYD- BASIC pipeline, we present a configurable reads- to- results analysis pipeline operated by non- expert users that greatly eases the investigation of potential outbreaks by expert end users. Results obtained with publicly available sequences show comparable performance to the original methods, while underlining the importance of standardized methods....
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