Annotation-Agnostic Differential Expression and Binding Analyses

My thesis work is centered around the development of R software packages for analyzing RNA sequencing (RNA-seq) and ChIP sequencing (ChIP-seq) high throughput genomic data. Chapter 2 describes the derfinder Bioconductor package which implements the DER Finder approach for identifying differentially expressed regions with RNA-seq data in an annotation-agnostic manner. Chapter 3 shows how derfinder can be applied to ChIP-seq data to identify differentially bounded regions. Chapter 4 describes the regionReport Bioconductor package for producing HTML or PDF reports from region-based genomic analyses, such as the derfinder analyses described in the previous chapters

Improving the value of public RNA-seq expression data by phenotype prediction.

Publicly available genomic data are a valuable resource for studying normal human variation and disease, but these data are often not well labeled or annotated. The lack of phenotype information for public genomic data severely limits their utility for addressing targeted biological questions. We develop an in silico phenotyping approach for predicting critical missing annotation directly from genomic measurements using well-annotated genomic and phenotypic data produced by consortia like TCGA and GTEx as training data. We apply in silico phenotyping to a set of 70 000 RNA-seq samples we recently processed on a common pipeline as part of the recount2 project. We use gene expression data to build and evaluate predictors for both biological phenotypes (sex, tissue, sample source) and experimental conditions (sequencing strategy). We demonstrate how these predictions can be used to study cross-sample properties of public genomic data, select genomic projects with specific characteristics, and perform downstream analyses using predicted phenotypes. The methods to perform phenotype prediction are available in the phenopredict R package and the predictions for recount2 are available from the recount R package. With data and phenotype information available for 70,000 human samples, expression data is available for use on a scale that was not previously feasible

Diferenciación de la respuesta in vitro de Phaseolus vulgaris cv. ‘ICA Pijao’ y cv. ‘Tío Canela- 75’ a altas temperaturas

 To develop an in vitro selection protocol, it is necessary to differentiate between tolerant and susceptible cultivars to the studied factor. The present work was carried out with the objective of selecting the temperature that allows to in vitro differentiating the response of the Phaseolus vulgaris cultivars 'ICA Pijao' (susceptible) and 'Tío Canela-75' (tolerant) to high temperatures. Different temperatures (30, 35, 40, 45 and 50 ºC) were applied to seeds of the two cultivars. In vitro seed germination percentage, radicle and plumule length, number of explants that formed callus and number of shoots per callus were evaluated. It was possible to differentiate between cv. ‘ICA Pijao’ (susceptible) and ‘Tío Canela- 75’ (tolerant) with the variables radicle length, plumule length, percentage of calli formed and number of regenerated shoots. It was selected 35 °C as the selective agent.Para desarrollar un protocolo de selección in vitro se hace necesario lograr la diferenciación entre cultivares tolerantes y susceptibles al factor estudiado. El presente trabajo se realizó con el objetivo de seleccionar la temperatura que permita diferenciar in vitro la respuesta de los cultivares de Phaseolus vulgaris ‘ICA Pijao’ (susceptible) y ‘Tío Canela- 75’  (tolerante) a altas temperaturas. Se aplicaron diferentes temperatura (30, 35, 40, 45 y 50 ºC) a semillas de los  dos cultivares. Se evaluó in vitro, el porcentaje de germinación de las semillas, longitud de la radícula y la plúmula, número de explantes que formaron callos y número de brotes por callo.  Se logró diferenciar entre el cv. ‘ICA Pijao’ (susceptible) y ‘Tío Canela- 75’ (Tolerante) con las variables longitud de la radícula, longitud de la plúmula, porcentaje de callos formados y número de brotes regenerados.  Se seleccionó 35 ºC como agente selectivo

Non-coding Class Switch Recombination-Related Transcription in Human Normal and Pathological Immune Responses

Antibody class switch recombination (CSR) to IgG, IgA, or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant gene, downstream of the VDJH exon. CSR depends on non-coding (CSRnc) transcription of donor Iμ and acceptor IH exons, located 5′ upstream of each CH coding gene. Although, our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce. We analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA, and the Sequence Read Archive using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as, coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iμ, Iγ3, and Iγ1 in humans in response to vaccination. We mapped IH transcription for the human IGH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called “immune privileged” tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased Iδ transcription in ileal mucosa in Crohn's disease with ulceration was found. In conclusion, CSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response (i.e., cytokine milieu) in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome

Efecto del tratamiento térmico a altas temperaturas sobre la germinación in vitro de semillas de Phaseolus vulgaris cv. ‘ICA Pijao’

The common bean (Phaseolus vulgaris L.) production is limited by biotic and abiotic factors. Among them, water stress, saline or high temperatures. To solve this problem, genetic improvement programs are aimed at the search for tolerant cultivars. The objective of this work was to determine the effect of heat treatment at high temperatures on the in vitro germination of P. vulgaris cv. ‘ICA Pijao’. For this, seeds of 1-4 months of harvest were used. It were treated at 35 ± 2 °C in an incubator or immersed in water in a thermal bath. As a control, seeds at room temperature (28 ± 2 °C) were used. The results showed that in vitro germination was not less than 90%, however the two heat treatment conditions affected the length and thickness of the radicle and the length of the plumule. Considering the results, the ease of application and the non-affectation of germination, it is recommended to use the heat treatment at 35 ± 2 ºC in an incubator. This could be used to induce high temperature stress in the common beans cv. ‘ICA Pijao’ in an in vitro selection protocol.La producción de frijol común (Phaseolus vulgaris L.) se limita por factores bióticos y abióticos. Entre ellos, el estrés hídrico, salino o por altas temperaturas. Para dar solución a esta problemática, los programas de mejoramiento genético se encaminan a la búsqueda de cultivares tolerantes. El objetivo de este trabajo fue determinar el efecto del tratamiento térmico a altas temperaturas sobre la germinación in vitro de P. vulgaris cv. ‘ICA Pijao’. Para ello, se utilizaron semillas de 1-4 meses de cosechadas. Estas fueron tratadas a 35±2 ºC en incubadora o sumergidas en agua en baño térmico. Como control se emplearon semillas a temperatura ambiente (28±2 ºC). Los resultados mostraron que la germinación in vitro no fue inferior al 90%, sin embargo las dos condiciones de tratamiento térmico afectaron la longitud y el grosor de la radícula y la longitud de la plúmula. Considerando los resultados, la facilidad de aplicación y la no afectación de la germinación se recomienda utilizar el tratamiento térmico a 35±2 ºC en incubadora.  Este podría emplearse para inducir estrés por altas temperaturas en el cultivar de frijol común ‘ICA Pijao’ en un protocolo de selección in vitro

Líneas de grano negro de Phaseolus vulgaris L. promisorias por respuesta a condiciones de estrés hídrico

Stress caused by drought affects food crops, such as common bean (Phaseolus vulgaris L.). The objective of this work was to determine the response in the greenhouse of black grain common beans lines in stress conditions for irrigation to pre-flowering. Four possible black grain mutant lines were evaluated supporting two treatments: application of irrigation considering the water requirements of the crop and the second, application of irrigation to pre-flowering. The sowing and the attention to the crop were carried out, with the indications established in the technical instructions. Ten plants were randomly selected in each treatment and evaluated: length of the roots/plants (cm), number of legumes/plant, number of grains/legumes and weight of 100 seeds (g). As a result, the lines showed the highest values of root length in the conditions of irrigation application to pre-flowering. In the rest of the variables evaluated, the lines showed a similar response in two treatments. This response indicated they have a certain tolerance to stress conditions.El estrés causado por la sequía afecta el rendimiento de cultivos de importancia alimentaria como el frijol común (Phaseolus vulgaris L.). El presente trabajo tuvo como objetivo determinar la respuesta de líneas de frijol común de grano negro en casa de cultivo con estrés hídrico hasta la prefloración. Se evaluaron cuatro líneas posibles mutantes de grano negro bajo dos tratamientos: aplicación de riego considerando los requerimientos hídricos del cultivo y aplicación de riego solo hasta la prefloración. La siembra y las atenciones al cultivo se realizaron en correspondencia con las indicaciones establecidas en el instructivo técnico. Se seleccionaron diez plantas al azar en cada tratamiento y se midió longitud de las raíces por planta (cm), se cuantificó el número de legumbres por planta y el número de granos por legumbre. Además, se determinó el peso de 100 semillas (g). Como resultados las líneas mostraron los mayores valores de longitud de las raíces en las condiciones de aplicación de riego hasta prefloración. El resto de las variables evaluadas fueron similares en ambos tratamientos, lo que indicó que poseen cierta tolerancia a estas condiciones de estrés hídrico

Efecto de radiaciones gamma 60Co sobre la regeneración de plantas de Phaseolus vulgaris L. cultivar 'ICA Pijao' vía organogénesis indirecta

In Phaseolus vulgaris L. breeding programs has been used mutation induction, however, no radiosensitivity studies have been performed in vitro in order to define the optimal dose of radiation to be applied. The aim of this study was to determine the effect of gamma radiation in the formation, multiplication of callus and plant regeneration of P.vulgaris cultivar 'ICA Pijao' via indirect organogenesis. Seeds were irradiated with doses of 70 to 100 Gy and then it placed to in vitro germinate. From these, calli were formed and were regenerated. The number of germinated seeds was quantified and root length was measured. Callus formation in the number of explants that formed calli were recorded and fresh weight was determined at 21 days of culture as in multiplication. In plant regeneration, the number of shoots per callus and the number of shoots with roots were quantified and coloration that they had described. The application of gamma radiation on seeds from the lowest dose, induced a reduction in the percentage of explants with callus formation below 50% compared to control unirradiated, fresh mass of the callus and the shoot regeneration were below 30% compared to control, which showed values higher than 70%. Keywords: beans, in vitro mutations, breeding, gamma radiation, seedsEn programas de mejoramiento genético de Phaseolus vulgaris L. se ha empleado la inducción de mutaciones, sin embargo, no se han realizado estudios de radiosensibilidad in vitro que permitan definir la dosis óptima de radiación a aplicar. El objetivo del presente trabajo fue determinar el efecto de radiaciones gamma en la formación, multiplicación de callos y la regeneración de plantas de Phaseolus vulgaris L. cultivar 'ICA Pijao' vía organogénesis indirecta. Se irradiaron semillas con dosis de 70 a 100 Gy que posteriormente se colocaron a germinar in vitro. A partir de estas se formaron callos y regeneraron plantas. Se cuantificó el número de semillas germinadas y se midió la longitud de las raíces. En la formación de callos se registró el número de explantes que formaron callos y se determinó su masa fresca a los 21 días de cultivo al igual que en la multiplicación. En la regeneración de plantas se cuantificó el número de brotes por callo, el número de brotes con raíces y se describió la coloración que estos presentaron. Desde las dosis más bajas, la aplicación de radiaciones gamma sobre semillas indujo una reducción en el porcentaje de explante con formación de callo, por debajo del 50% en comparación con el control sin irradiar, la masa fresca de los callos, así como la regeneración de brotes, que estuvo por debajo del 30% en comparación con el control, que mostró valores superiores al 70%. Palabras clave: frijol, mutaciones in vitro, mejoramiento genético, semilla