RNA‐seq: Applications and Best Practices

RNA‐sequencing (RNA‐seq) is the state‐of‐the‐art technique for transcriptome analysis that takes advantage of high‐throughput next‐generation sequencing. Although being a powerful approach, RNA‐seq imposes major challenges throughout its steps with numerous caveats. There are currently many experimental options available, and a complete comprehension of each step is critical to make right decisions and avoid getting into inconclusive results. A complete workflow consists of: (1) experimental design; (2) sample and library preparation; (3) sequencing; and (4) data analysis. RNA‐seq enables a wide range of applications such as the discovery of novel genes, gene/transcript quantification, and differential expression and functional analysis. This chapter will encompass the main aspects from sample preparation to downstream data analysis. It will be discussed how to obtain high‐quality samples, replicates amount, library preparation, sequencing platforms and coverage, focusing on best recommended practices based on specialized literature. Basic techniques and well‐known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments

CALANGO: a phylogeny-aware comparative genomics tool for discovering quantitative genotype-phenotype associations across species

Living species vary significantly in phenotype and genomic content. Sophisticated statistical methods linking genes with phenotypes within a species have led to breakthroughs in complex genetic diseases and genetic breeding. Despite the abundance of genomic and phenotypic data available for thousands of species, finding genotype-phenotype associations across species is challenging due to the non-independence of species data resulting from common ancestry. To address this, we present CALANGO (comparative analysis with annotation-based genomic components), a phylogeny-aware comparative genomics tool to find homologous regions and biological roles associated with quantitative phenotypes across species. In two case studies, CALANGO identified both known and previously unidentified genotype-phenotype associations. The first study revealed unknown aspects of the ecological interaction between Escherichia coli, its integrated bacteriophages, and the pathogenicity phenotype. The second identified an association between maximum height in angiosperms and the expansion of a reproductive mechanism that prevents inbreeding and increases genetic diversity, with implications for conservation biology and agriculture

FC-R2: Um atlas completo de expressão de ARNs não codificadores utilizando um pipeline padronizado

Submitted by Eddie Imada ([email protected]) on 2019-09-11T18:20:02Z No. of bitstreams: 1 Final.pdf: 9401729 bytes, checksum: 09211c448aa44290f69b06e98335b3dc (MD5)Made available in DSpace on 2019-09-11T18:20:02Z (GMT). No. of bitstreams: 1 Final.pdf: 9401729 bytes, checksum: 09211c448aa44290f69b06e98335b3dc (MD5) Previous issue date: 2019-05-29CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas GeraisCAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorIn recent years, in depth exploration of genomes structure and function has revealed a central role for non-coding RNAs (ncRNAs) in orchestrating key biological and cellular processes through the fine tuning of gene expression regulation. Most importantly, the understanding of the role for ncRNAs has also started to emerge in human disease pathogenesis. This further speaks to the importance of an in-depth characterization of ncRNA involvement in diseases, including cancer. In this work, we have built a comprehensive atlas of gene expression, named FC-R2, across the human transcriptome containing over 100,000 genes by leveraging two publicly available resources: the FANTOM CAGE Associated Transcriptome (FANTOM-CAT), and recount2. The FANTOM-CAT is a comprehensive meta-assembly of the human transcriptome encompassing coding and non-coding genes, including promoters, enhancers, and lncRNAs. recount2 is the largest, available collection of human RNA-seq data processed and quantified using a unified pipeline, containing over 4.4 trillion reads from over 70,000 human samples from the SRA, GTEx and TCGA projects. Using FC-R2 gene expression summaries across human tissue samples from the GTEx project, we validated our approach by reproducing key findings recently described by the FANTOM consortium and the TCGA Pan-Cancer atlas. We also demonstrated the power and usability of the FC-R2 by performing two case studies in prostate cancer highlighting potential “novel” lncRNAs players involved in the clinically relevant prostate cancer phenotype. Finally, we make the FC-R2 atlas available as a public tool to empower other researchers to study important biological and clinical phenotypes and identify new candidate ncRNAs for further investigation.Recentemente, estudos à fundo das funções e estrutura de genomas revelou que RNAs não codificadores desempenham um papel essencial no controle e regulação de processos biológicos e celulares através da regulação da expressão gênica. Estes mecanismos também começaram a ser elucidados em doenças humanas, destacando a importância da caracterização dos papéis desempenhados pelos RNAs não-codificadores em doenças, como o câncer. Neste trabalho, nós construímos um atlas de expressão gênica do transcriptoma humano contendo mais de 100.000 genes fazendo uso de dois recursos públicos: o transcriptoma associado à CAGE do projeto FANTOM (do inglês FANTOM-CAT) e o recount2, denominado FC-R2. O FANTOM-CAT é uma meta-montagem completa do transcriptoma humano contendo ambos genes codificadores e não-codificadores, incluindo promotores, enhancers e RNAs não codificadores longos. Recount2 é a maior coleção disponível de dados de RNA-seq humano processados e quantificados utilizando um pipeline unificado contendo mais de 4,4 trilhões de bases e mais de 70.000 amostras humanas derivadas do SRA e dos projetos TCGA e GTEx. Utilizando dados do GTEx derivados do FC-R2, nós validamos nossa abordagem ao reproduzir diversas descobertas importantes descritas recentemente pelo projeto FANTOM e do Pan-cancer atlas do TCGA. Em dois estudos de caso, nós também demonstramos a utilidade e capacidade do FC-R2 em recuperar novos RNAs não-codificadores longos potencialmente envolvidos em fenótipos de importância clínica. Concluindo, nós disponibilizamos o atlas FC-R2 como uma ferramenta publica para permitir que outros pesquisadores sejam capazes de identificar novos RNAs não-codificadores em fenótipos de interesse

Influenza Vaccination and COVID-19 Mortality in the USA: An Ecological Study

The COVID-19 mortality rate is higher in the elderly and in those with pre-existing chronic medical conditions. The elderly also suffer from increased morbidity and mortality from seasonal influenza infections; thus, an annual influenza vaccination is recommended for them. In this study, we explore a possible county-level association between influenza vaccination coverage in people aged 65 years and older and the number of deaths from COVID-19. To this end, we used COVID-19 data up to 14 December 2020 and US population health data at the county level. We fit quasi-Poisson regression models using influenza vaccination coverage in the elderly population as the independent variable and the COVID-19 mortality rate as the outcome variable. We adjusted for an array of potential confounders using different propensity score regression methods. Results show that, on the county level, influenza vaccination coverage in the elderly population is negatively associated with mortality from COVID-19, using different methodologies for confounding adjustment. These findings point to the need for studying the relationship between influenza vaccination and COVID-19 mortality at the individual level to investigate any underlying biological mechanisms

CALANGO: A phylogeny-aware comparative genomics tool for discovering quantitative genotype-phenotype associations across species

Living species vary significantly in phenotype and genomic content. Sophisticated statistical methods linking genes with phenotypes within a species have led to breakthroughs in complex genetic diseases and genetic breeding. Despite the abundance of genomic and phenotypic data available for thousands of species, finding genotype-phenotype associations across species is challenging due to the non-independence of species data resulting from common ancestry. To address this, we present CALANGO (comparative analysis with annotation-based genomic components), a phylogeny-aware comparative genomics tool to find homologous regions and biological roles associated with quantitative phenotypes across species. In two case studies, CALANGO identified both known and previously unidentified genotype-phenotype associations. The first study revealed unknown aspects of the ecological interaction between Escherichia coli, its integrated bacteriophages, and the pathogenicity phenotype. The second identified an association between maximum height in angiosperms and the expansion of a reproductive mechanism that prevents inbreeding and increases genetic diversity, with implications for conservation biology and agriculture

Identification of genes involved in indole-3-acetic acid biosynthesis by Gluconacetobacter diazotrophicus PAL5 strain using transposon mutagenesis

Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds that the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao, cccA and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and provides evidence for the involvement of an L-amino acid oxidase gene cluster in the biosynthesis of IAA. Furthermore, we showed that the mutant strains with reduction in IAA biosynthesis ability, in consequence of the lower transcription levels of genes of the lao cluster, had remarkable effects in development of rice roots

Donor-derived acute myeloid leukemia in solid organ transplantation

We report the transmission of acute myeloid leukemia (AML) undetected at donation from a deceased organ donor to two kidneys and one liver recipients. We reviewed the medical records, and performed molecular analyses and whole exome sequencing (WES) to ascertain AML donor origin and its molecular evolution. The liver recipient was diagnosed 11 months after transplantation and died from complications 2 months later. The two kidney recipients (R1 and R2) were diagnosed 19 and 20 months after transplantation and both received treatment for leukemia. R1 died of complications 11 months after diagnosis, while R2 went into complete remission for 44months, before relapsing. R2 died 10 months later of complications from allogenic bone marrow transplantation. Microsatellite analysis demonstrated donor chimerism in circulating cells from both kidney recipients. Targeted molecular analyses and medical records revealed NPM1 mutation present in the donor and recipients, while FLT3 was mutated only in R1. These findings were confirmed by WES, which revealed additional founder and clonal mutations, and HLA genomic loss in R2. In conclusion, we report the first in--depth genomic analysis of AML transmission following solid organ transplantation, revealing distinct clonal evolution, and providing a potential molecular explanation for tumor escape