1,224 research outputs found
Reconstructing DNA copy number by joint segmentation of multiple sequences
The variation in DNA copy number carries information on the modalities of
genome evolution and misregulation of DNA replication in cancer cells; its
study can be helpful to localize tumor suppressor genes, distinguish different
populations of cancerous cell, as well identify genomic variations responsible
for disease phenotypes. A number of different high throughput technologies can
be used to identify copy number variable sites, and the literature documents
multiple effective algorithms. We focus here on the specific problem of
detecting regions where variation in copy number is relatively common in the
sample at hand: this encompasses the cases of copy number polymorphisms,
related samples, technical replicates, and cancerous sub-populations from the
same individual. We present an algorithm based on regularization approaches
with significant computational advantages and competitive accuracy. We
illustrate its applicability with simulated and real data sets.Comment: 54 pages, 5 figure
Comparative genomics of recent adaptation in Candida pathogens
[eng] Fungal infections pose a serious health threat, affecting >1,000 million people and causing ~1.5 million deaths each year. The problem is growing due to insufficient diagnostic and therapeutic options, increased number of susceptible patients, expansion of pathogens partly linked to climate change and the rise of antifungal drug resistance. Among other fungal pathogens, Candida species are a major cause of severe hospital-acquired infections, with high mortality in immunocompromised patients. Various Candida pathogens constitute a public health issue, which require further efforts to develop new drugs, optimize currently available treatments and improve diagnostics. Given the high dynamism of Candida genomes, a promising strategy to improve current therapies and diagnostics is to understand the evolutionary mechanisms of adaptation to antifungal drugs and to the human host. Previous work using in vitro evolution, population genomics, selection inferences and Genome Wide Association Studies (GWAS) have partially clarified such recent adaptation, but various open questions remain. In the three research articles that conform this PhD thesis we addressed some of these gaps from the perspective of comparative genomics.
First, we addressed methodological issues regarding the analysis of Candida genomes. Studying recent adaptation in these pathogens requires adequate bioinformatic tools for variant calling, filtering and functional annotation. Among other reasons, current methods are suboptimal due to limited accuracy to identify structural variants from short read sequencing data. In addition, there is a need for easy-to-use, reproducible variant calling pipelines. To address these gaps we developed the “personalized Structural Variation detection” pipeline (perSVade), a framework to call, filter and annotate several variant types, including structural variants, directly from reads. PerSVade enables accurate identification of structural variants in any species of interest, such as Candida pathogens. In addition, our tool automatically predicts the structural variant calling accuracy on simulated genomes, which informs about the reliability of the calling process. Furthermore, perSVade can be used to analyze single nucleotide polymorphisms and copy number-variants, so that it facilitates multi-variant, reproducible genomic studies. This tool will likely boost variant analyses in Candida pathogens and beyond.
Second, we addressed open questions about recent adaptation in Candida, using perSVade for variant identification. On the one hand, we investigated the evolutionary mechanisms of drug resistance in Candida glabrata. For this, we used a large-scale in vitro evolution experiment to study adaptation to two commonly-used antifungals: fluconazole and anidulafungin. Our results show rapid adaptation to one or both drugs, with moderate fitness costs and through few mutations in a narrow set of genes. In addition, we characterize a novel role of ERG3 mutations in cross-resistance towards fluconazole in
anidulafungin-adapted strains. These findings illuminate the mutational paths leading to drug resistance and cross-resistance in Candida pathogens. On the other hand, we reanalyzed ~2,000 public genomes and phenotypes to understand the signs of recent selection and drug resistance in six major Candida species: C. auris, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis and C. orthopsilosis. We found hundreds of genes under recent selection, suggesting that clinical adaptation is diverse and complex. These involve species-specific but also convergently affected processes, such as cell adhesion, which could underlie conserved adaptive mechanisms. In addition, using GWAS we predicted known drivers of antifungal resistance alongside potentially novel players. Furthermore, our analyses reveal an important role of generally-overlooked structural variants, and suggest an unexpected involvement of (para)sexual recombination in the spread of resistance. Taken together, our findings provide novel insights on how Candida pathogens adapt to human-related environments and suggest candidate genes that deserve future attention. In summary, the results of this thesis improve our knowledge about the mechanisms of recent adaptation in Candida pathogens, which may enable improved therapeutic and diagnostic applications.[cat] Les infeccions fúngiques representen una greu amenaça per a la salut, afectant a més de 1.000 milions de persones i causant aproximadament 1,5 milions de morts cada any. El problema està augmentant a causa d’unes opcions terapèutiques i diagnòstiques insuficients, l'increment del nombre de pacients susceptibles, l'expansió dels patògens parcialment vinculada al canvi climàtic i l'augment de la resistència als fàrmacs antifúngics. D’entre diversos fongs patògens, els llevats del gènere Candida són una causa important d'infeccions nosocomials, amb una alta mortalitat en pacients immunodeprimits. Diverses espècies de Candida constitueixen un problema de salut pública, cosa que requereix més esforços per a desenvolupar nous medicaments, optimitzar els tractaments disponibles i millorar els diagnòstics. Tenint en compte el dinamisme genòmic d’aquests patògens, una estratègia prometedora per millorar les teràpies i diagnòstics actuals és comprendre els mecanismes evolutius d'adaptació als fàrmacs antifúngics i a l’hoste humà. Treballs anteriors utilitzant l'evolució in vitro, la genòmica de poblacions, les inferències de selecció i els estudis d'associació de genoma complet (GWAS, per les sigles en anglès) han aclarit parcialment aquesta adaptació recent, però encara hi ha diverses preguntes obertes. En els tres articles que conformen aquesta tesi doctoral, hem abordat algunes d'aquestes preguntes des de la perspectiva de la genòmica comparativa.
En primer lloc, hem abordat qüestions metodològiques relatives a l'anàlisi dels genomes de les espècies Candida. L'estudi de l'adaptació recent en aquests patògens requereix eines bioinformàtiques adequades per a la detecció, filtratge i anotació funcional de variants genètiques. Entre altres raons, els mètodes actuals són subòptims a causa de la limitada precisió per identificar variants estructurals a partir de dades de seqüenciació amb lectures curtes. A més, hi ha una necessitat d’eines computacionals per a la detecció de variants que siguin senzilles d'utilitzar i reproduibles. Per abordar aquestes mancances, hem desenvolupat el mètode bioinformàtic "personalized Structural Variation detection" (perSVade), una eina que permet la detecció, filtratge i anotació de diversos tipus de variants, incloent-hi les variants estructurals, directament des de les lectures. PerSVade permet la identificació precisa de les variants estructurals en qualsevol espècie d'interès, com ara els patògens Candida. A més, la nostra eina prediu automàticament la precisió de la detecció d’aquestes variants en genomes simulats, la qual cosa informa sobre la fiabilitat del procés. Finalment, perSVade es pot utilitzar per analitzar altres tipus de variants, com els polimorfismes de nucleòtid únic o els canvis en el nombre de còpies, facilitant així estudis genòmics integrals i reproduibles. Aquesta eina probablement impulsarà les anàlisis genòmiques en els patògens Candida i també en altres espècies.
En segon lloc, hem abordat algunes de les preguntes obertes sobre l'adaptació recent en els llevats Candida, utilitzant perSVade per a la identificació de variants. D'una banda, hem investigat els mecanismes evolutius de resistència als fàrmacs antifúngics en Candida glabrata. Per a això, hem utilitzat un experiment
d'evolució in vitro a gran escala per estudiar l'adaptació a dos antifúngics comuns: el fluconazol i l’anidulafungina. Els nostres resultats mostren una adaptació ràpida a un o ambdós fàrmacs, amb un cost per al creixement moderat i a través de poques mutacions en un nombre reduït de gens. A més, hem caracteritzat un paper nou de les mutacions en ERG3 en la resistència creuada al fluconazol en soques adaptades a anidulafungina. Aquests descobriments aclareixen els processos mutacionals que condueixen a la resistència als fàrmacs i a la resistència creuada en els patògens Candida. D'altra banda, hem re-analitzat aproximadament 2.000 genomes i fenotips disponibles en repositoris públics per a comprendre els senyals genòmics de selecció recent i de resistència a fàrmacs antifúngics, en sis espècies rellevants de Candida: C. auris, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis i C. orthopsilosis. Hem trobat centenars de gens sota selecció recent, suggerint que l'adaptació clínica és diversa i complexa. Aquests gens estan relacionats amb funcions específiques de cada espècie, però també trobem processos alterats de manera similar en diferents patògens, com per exemple l’adhesió cel·lular, cosa que indica fenòmens d’adaptació conservats. A part, utilitzant GWAS hem predit mecanismes esperats de resistència a antifúngics i també possibles nous factors. A més, les nostres anàlisis revelen un paper important de les variants estructurals, generalment poc estudiades, i suggereixen una implicació inesperada de la recombinació (para)sexual en la propagació de la resistència. En conjunt, els nostres descobriments proporcionen noves perspectives sobre com els patògens Candida s'adapten als entorns humans, i suggereixen gens candidats que mereixen investigacions futures. En resum, els resultats d’aquesta tesi milloren el nostre coneixement sobre els mecanismes d'adaptació recent en els patògens Candida, cosa que pot permetre el disseny de noves teràpies i diagnòstics
A draft human pangenome reference
Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individual
Computational approaches for improving treatment and prevention of viral infections
The treatment of infections with HIV or HCV is challenging. Thus, novel drugs and new computational approaches that support the selection of therapies are required. This work presents methods that support therapy selection as well as methods that advance novel antiviral treatments. geno2pheno[ngs-freq] identifies drug resistance from HIV-1 or HCV samples that were subjected to next-generation sequencing by interpreting their sequences either via support vector machines or a rules-based approach. geno2pheno[coreceptor-hiv2] determines the coreceptor that is used for viral cell entry by analyzing a segment of the HIV-2 surface protein with a support vector machine. openPrimeR is capable of finding optimal combinations of primers for multiplex polymerase chain reaction by solving a set cover problem and accessing a new logistic regression model for determining amplification events arising from polymerase chain reaction. geno2pheno[ngs-freq] and geno2pheno[coreceptor-hiv2] enable the personalization of antiviral treatments and support clinical decision making. The application of openPrimeR on human immunoglobulin sequences has resulted in novel primer sets that improve the isolation of broadly neutralizing antibodies against HIV-1. The methods that were developed in this work thus constitute important contributions towards improving the prevention and treatment of viral infectious diseases.Die Behandlung von HIV- oder HCV-Infektionen ist herausfordernd. Daher werden neue Wirkstoffe, sowie neue computerbasierte Verfahren benötigt, welche die Therapie verbessern. In dieser Arbeit wurden Methoden zur Unterstützung der Therapieauswahl entwickelt, aber auch solche, welche neuartige Therapien vorantreiben. geno2pheno[ngs-freq] bestimmt, ob Resistenzen gegen Medikamente vorliegen, indem es Hochdurchsatzsequenzierungsdaten von HIV-1 oder HCV Proben mittels Support Vector Machines oder einem regelbasierten Ansatz interpretiert. geno2pheno[coreceptor-hiv2] bestimmt den HIV-2 Korezeptorgebrauch dadurch, dass es einen Abschnitt des viralen Oberflächenproteins mit einer Support Vector Machine analysiert. openPrimeR kann optimale Kombinationen von Primern für die Multiplex-Polymerasekettenreaktion finden, indem es ein Mengenüberdeckungsproblem löst und auf ein neues logistisches Regressionsmodell für die Vorhersage von Amplifizierungsereignissen zurückgreift. geno2pheno[ngs-freq] und geno2pheno[coreceptor-hiv2] ermöglichen die Personalisierung antiviraler Therapien und unterstützen die klinische Entscheidungsfindung. Durch den Einsatz von openPrimeR auf humanen Immunoglobulinsequenzen konnten Primersätze generiert werden, welche die Isolierung von breit neutralisierenden Antikörpern gegen HIV-1 verbessern. Die in dieser Arbeit entwickelten Methoden leisten somit einen wichtigen Beitrag zur Verbesserung der Prävention und Therapie viraler Infektionskrankheiten
Integrative Transcriptomic Analysis of Long Intergenic Non-Coding RNAs in Cancer.
Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017
Recommended from our members
A tool of "barcoded viruses" to study influenza virus transmission dynamics
The aim of this study was to establish a novel version of powerful “barcode viruses” as a
tool for studying the replication and transmission dynamics of influenza virus in vitro
and in vivo.
Five barcoded APR8 viruses were firstly used to investigate infection kinetics (e.g.
single- and multi-hit events, particle clumping and temporal aspects of co-infection) in
vitro. This work demonstrated that the majority of infectious events in cell culture were
single-hit events, but a significant number of infections were initiated by more than one
virus particles (consistent with virus aggregation during release). Reassortment was
found to occur efficiently and ubiquitously when near-isogenic viruses co-infected cells.
The timing of asynchronous co-infection revealed that super-infection was possible if
the second virus encountered the cell within 4 hr of the first virus. The super-infecting
virus showed accelerated replication and enhanced yield, suggesting the second virus
can take advantage of the already initiated replication machinery. Beyond this time
point (coincident with the onset of progeny release from the first virus) the second virus
was blocked by the initial infecting viruses.
Five virus libraries carrying ~2000 individually identifiable variants were then
generated for in vivo study. Amplification of the viral libraries in Madin-Darby canine
kidney (MDCK) cells was achieved without substantial bottlenecking or preferential
selection of specific sequences. Thirdly, two pilot studies in pigs demonstrated that
intranasal inoculation resulted in substantial bottlenecking and a relatively small
proportion of the inoculum gave rise to productive infection. Consequently, distinct viral
populations were found in different nostrils and could persist over the course of the
infection due to anatomical partitioning. Distinct sub-populations could be distinguished
in other tissue sites (e.g. trachea and lung). Super-infection of individual pigs could
occur around 2 days following primary exposure. The identity of the donor pigs could be
determined by the barcode identities. In the first pilot study, around 600 variants were
seen in each donor pig directly inoculated with approximately 6000 variants of the
barcoded viruses. When a pig was co-housed with 3 donors, a typical transmission dose
of 73-151 variants were seen. To further study the transmission dose between a single
donor and recipient, a transmission dose defined as 30-60 on 2 days post contact (d.p.c)
and 20-50 on 3 d.p.c was observed.
To conclude, my PhD project has developed a powerful tool with a wide range of
applications in influenza biology, particularly for studying transmission dynamics in a
natural host system
On the Analysis of DNA Methylation
Recent genome-wide studies lend support to the idea that the patterns of DNA methylation are in some way related either causally or as a readout of cell-type specific protein binding. We lay the groundwork for a framework to test whether the pattern of DNA methylation levels in a cell combined with protein binding models is sufficient to completely describe the location of the component of proteins binding to its genome in an assayed context. There is only one method, whole-genome bisulfite sequencing, WGBS, available to study DNA methylation genome-wide at such high resolution, however its accuracy has not been determined on the scale of individual binding locations. We address this with a two-fold approach. First, we developed an alternative high-resolution, whole-genome assay using a combination of an enrichment-based and a restriction-enzyme-based assay of methylation, methylCRF. While both assays are considered inferior to WGBS, by using two distinct assays, this method has the advantage that each assay in part cancels out the biases of the other. Additionally, this method is up to 15 times lower in cost than WGBS. By formulating the estimation of methylation from the two methods as a structured prediction problem using a conditional random field, this work will also address the general problem of incorporating data of varying qualities -a common characteristic of biological data- for the purpose of prediction. We show that methylCRF is concordant with WGBS within the range of two WGBS methylomes. Due to the lower cost, we were able to analyze at high-resolution, methylation across more cell-types than previously possible and estimate that 28% of CpGs, in regions comprising 11% of the genome, show variable methylation and are enriched in regulatory regions. Secondly, we show that WGBS has inherent resulution limitations in a read count dependent manner and that the identification of unmethylated regions is highly affected by GC-bias in the underlying protocol suggesting simple estimate procedures may not be sufficient for high-resolution analysis. To address this, we propose a novel approach to DNA methylation analysis using change point detection instead of estimating methylation level directly. However, we show that current change-point detection methods are not robust to methylation signal, we therefore explore how to extend current non-parametric methods to simultaneously find change-points as well as characteristic methylation levels. We believe this framework may have the power to examine the connection between changes in methylation and transcription factor binding in the context of cell-type specific behaviors
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