Biology subjects

Vale-Silva, L. A.

Publications and source records attributed to Vale-Silva, L. A..

6 recordsLinked to original sources

bioRxiv2025

iss-nf: A Nextflow-based end-to-end in situ sequencing decoding workflow

In situ sequencing (ISS) offers a powerful approach for spatially resolved gene expression profiling within tissue samples, but the complexity of analyzing the resulting data has limited its broader use. Here, we present iss-nf, a Nextflow-based, end-to-end workflow designed to streamline the decoding of large ISS datasets. The workflow automates critical steps, including image registration, fluorescent spot detection, transcript decoding, and quality control (QC), offering a scalable, reproducible, and user-friendly solution for ISS data analysis. We successfully applied iss-nf on multiple large datasets, including publicly available mouse brain and breast cancer tissue datasets, as well as an in-house non-small cell lung cancer (NSCLC) ISS dataset. The workflow is designed to be accessible for both experienced researchers, as well as newcomers to spatial transcriptomics, providing a robust tool for analyzing large-scale ISS data. Our results suggest that iss-nf is a valuable contribution to the growing field of spatial transcriptomics, enabling precise, modular, reproducible, and, by means of automated tiling and parallelization, scalable analysis of tissue-specific gene expression.

bioinformatics↗

bioRxiv2020

A cohesin-independent mechanism modulates recombination activity along meiotic chromosomes

Successful meiotic recombination, and thus fertility, depends on conserved axis proteins that organize chromosomes into arrays of anchored chromatin loops and provide a protected environment for DNA exchange. Here, we show that the stereotypic chromosomal distribution of axis proteins in S. cerevisiae is the additive result of two independent pathways: a cohesin-dependent pathway, which was previously identified and mediates focal enrichment of axis proteins at gene ends, and a parallel cohesin-independent pathway that recruits axis proteins to broad genomic islands with high gene density. These islands exhibit elevated markers of crossover recombination as well as increased nucleosome density, which we show is a direct consequence of the underlying DNA sequence. A predicted PHD domain in the center of the axis factor Hop1 specifically mediates cohesin-independent axis recruitment. Intriguingly, other chromosome organizers, including cohesin, condensin, and topoisomerases, are differentially depleted from the same regions even in non-meiotic cells, indicating that these DNA sequence-defined chromatin islands exert a general influence on the patterning of chromosome structure.

genomics↗

bioRxiv2019

Topoisomerases modulate the timing of meiotic DNA breakage and repair

During meiotic prophase, concurrent transcription, recombination, and chromosome synapsis, place substantial topological strain on chromosomal DNA, but the role of topoisomerases in this context remains poorly defined. Here, we analyzed the roles topoisomerases I and II (Top1 and Top2) during meiotic prophase in Saccharomyces cerevisiae. We show that both topoisomerases accumulate primarily in promoter-containing intergenic regions of actively transcribing genes. Enrichment partially overlaps meiotic double-strand break (DSB) hotspots, but disruption of either topoisomerase has different effects during meiotic recombination. TOP1 disruption delays DSB induction and shortens the window of DSB accumulation by an unknown mechanism. By contrast, temperature-sensitive top2-1 mutants accumulate DSBs on synapsed chromosomes and exhibit a marked delay in meiotic chromosome remodeling. This defect results from a delay in recruiting the meiotic chromosome remodeler Pch2/TRIP13 but, unexpectedly, is not due to a loss of Top2 catalytic activity. Instead, mutant Top2-1 protein has reduced contact with chromatin but remains associated with meiotic chromosomes, and we provide evidence that this altered binding is responsible for the delay in chromosome remodeling. Our results imply independent roles for topoisomerases I and II in modulating meiotic recombination.

genomics↗

bioRxiv2018

SNP-ChIP: A versatile and tag-free method to quantify changes in protein binding across the genome

Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) is the method of choice for mapping genome-wide binding of chromatin-associated factors. However, broadly applicable methods for between-sample comparisons are lacking. Here, we introduce SNP-ChIP, a method that leverages small-scale intra-species polymorphisms, mainly SNPs, for quantitative spike-in normalization of ChIP-seq results. Sourcing spike-in material from the same species ensures antibody cross-reactivity and physiological coherence, thereby eliminating two central limitations of traditional spike-in approaches. We show that SNP-ChIP is robust to changes in sequencing depth and spike-in proportions, and reliably identifies changes in overall protein levels, irrespective of changes in binding distribution. Application of SNP-ChIP to test cases from budding yeast meiosis allowed discovery of novel regulators of the chromosomal protein Red1 and quantitative analysis of the DNA-damage associated histone modification{gamma} -H2AX. SNP-ChIP is fully compatible with the intra-species diversity of humans and most model organisms and thus offers a general method for normalizing ChIP-seq results.

genomics↗

bioRxiv2018

Synthetic chromosome fusion: effects on genome structure and function

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

synthetic biology↗

bioRxiv2017

Comparative Genomics Of Two Sequential Candida glabrata Clinical Isolates

Candida glabrata is an important fungal pathogen which develops rapidly antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator PDR1 result in upregulation of the transporters. In addition, we showed that the PDR1 mutations can contribute to enhance virulence in animal models. We were interested in this study to compare genomes of two specific C. glabrata related isolates, one of which was azole-susceptible (DSY562) while the other was azole-resistant (DSY565). DSY565 contained a PDR1 mutation (L280F) and was isolated after a time lapse of 50 days of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacbBio technology used here yielded 14 major contigs (sizes 0.18 Mb-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 contained each a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (66) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 non-synonymous SNPs (among which the expected PDR1 mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes were containing a DNA mismatch repair allele of MSH2 known to be involved in the so-called hypermutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a MSH2 defect. In conclusion, this study is the first to compare genomes of C. glabrata sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points were exhibiting limited variations, even if submitted to the host pressure.

genomics↗