Biology subjects

de Paula Baptista, R.

Publications and source records attributed to de Paula Baptista, R..

7 recordsLinked to original sources

bioRxiv2024

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA (<40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of Cryptosporidium species that have been found to infect humans (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage, and facilitates analyses of genetic variants in many samples, while decreasing overall costs.

genomics↗

bioRxiv2024

The repetitive genome of the Ixodes ricinus tick reveals transposable elements have driven genome evolution in ticks

Ticks are obligate blood-feeding parasites associated with a huge diversity of diseases globally. The hard tick Ixodes ricinus is the key vector of Lyme borreliosis and tick-borne encephalitis in Western Eurasia. Ixodes ticks have large and repetitive genomes that are not yet well characterized. Here we generate two high-quality I. ricinus genome assemblies, with haploid genome sizes of approximately 2.15 Gbp. We find transposable elements comprise at least 69% of the two I. ricinus genomes, amongst the highest proportions found in animals. The transposable elements in ticks are highly diverse and novel, so we constructed a repeat library for ticks using our I. ricinus genomes and the genome of I. scapularis, another major tick vector of Lyme borreliosis. To understand the impact of transposable elements on tick genomes we compared their accumulation in the two Ixodes sister species. We find transposable elements in these two species to be drivers of genome evolution in ticks. The I. ricinus genome assemblies and our tick repeat library will be valuable resources for biological insights into this important ectoparasite. Our findings highlight that further research into the impact of transposable elements on the genomes of blood-feeding parasites is required.

genomics↗

bioRxiv2024

Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing

Multiple Displacement Amplification (MDA) outperforms conventional PCR in long fragment and whole genome amplification which makes it attractive to couple with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for genome sequence assembly using Oxford Nanopore Technologies (ONT) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with the ability to generate high-quality data from samples starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size-increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

bioinformatics↗

bioRxiv2024

Cefiderocol heteroresistance associated with mutations in TonB-dependent receptor genes in Pseudomonas aeruginosa of clinical origin

The siderophore-cephalosporin cefiderocol(FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR, pirS, pirA, piuA or piuD from 498 unique isolates collected before the introduction of FDC from 4 clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n=15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

microbiology↗

bioRxiv2023

Genetic crosses within and between species of Cryptosporidium

Parasites and their hosts are engaged in rapid coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing C. parvum, a parasite of cattle and humans, and C. tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity. Significance statementThe parasite Cryptosporidium is a leading cause of diarrheal disease. While infection is common all around the world, young children experiencing malnutrition are impacted most profoundly, and the disease is an important contributor to early childhood mortality. This study experimentally demonstrates that different strains and even species of Cryptosporidium can recombine their genomes through sex. The progeny of such genetic crosses shows combined features of both parents, with resistance to multiple drugs being one example. Sex thus provides a critical mechanism for the parasite to rapidly adapt to changing environments and hosts. Genetic crosses as an experimental tool may also be harnessed in the future to discover the genes underlying differences in virulence, drug sensitivity, and immunogenicity between parasite isolates.

microbiology↗

bioRxiv2023

New T2T assembly of Cryptosporidium parvum IOWA annotated with reference genome gene identifiers

Cryptosporidium parvum is a significant pathogen causing gastrointestinal infections in humans and animals, that is spread through the ingestion of contaminated food and water. Despite its global impact on public health, generating a C. parvum genome sequence has always been challenging due to a lack of in vitro cultivation systems and challenging sub-telomeric gene families. A gapless telomere to telomere genome assembly has been created for Cryptosporidium parvum IOWA obtained from Bunch Grass Farms, named here as CpBGF. There are 8 chromosomes that total 9,259,183 bp. The new hybrid assembly which was generated with Illumina and Oxford Nanopore resolves complex sub-telomeric regions of chromosomes 1, 7 and 8. To facilitate ease of use and consistency with the literature, whenever possible, chromosomes have been oriented and genes in this annotation have been given the same gene IDs used in the current reference genome sequence generated in 2004. The annotation of this assembly utilized considerable RNA expression evidence, thus, untranslated regions, long noncoding RNAs and antisense RNAs are annotated. The CpBGF genome assembly serves as a valuable resource for understanding the biology, pathogenesis, and transmission of C. parvum, and it facilitates the development of diagnostics, drugs, and vaccines against cryptosporidiosis.

genomics↗

bioRxiv2022

Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA

Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in clinical bacterial isolates. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria, we compared different approaches to identify best practices for detection of AMR genes, including: total genomic DNA and plasmid DNA extractions, solo assembly of Illumina short-reads and of ONT long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA indicating that, at least in these three enterobacterial genera, purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. We also found that Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility corresponded well with genotypes identified by sequencing, but the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS to expedite patient treatment and to track AMR genes within the hospital and community.

microbiology↗