High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Author(s)

Aquime Goncalves, Andre Nicolau ; Maso, Vanessa Escolano ; Santos de Castro, Icaro Maia ; Vasconcelos, Amanda Pereira ; Tomio Ogava, Rodrigo Luiz ; Nakaya, Helder, I

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 181, p. 22-pg., 2022-03-01.

Abstract

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of the disease. To study such processes, researchers can use high-throughput sequencing techniques (RNA-seq) that measure the dynamic changes of the host transcriptome at different stages of infection, clinical outcomes, or disease severity.This investigation can lead to a better understanding of the diseases, as well as uncovering potential drug targets and treatments. The protocol presented here describes a complete pipeline to analyze RNA-sequencing data from raw reads to functional analysis. The pipeline is divided into five steps: (1) quality control of the data; (2) mapping and annotation of genes; (3) statistical analysis to identify differentially expressed genes and co-expressed genes; (4) determination of the molecular degree of the perturbation of samples; and (5) functional analysis. Step 1 removes technical artifacts that may impact the quality of downstream analyses. In step 2, genes are mapped and annotated according to standard library protocols. The statistical analysis in step 3 identifies genes that are differentially expressed or co-expressed in infected samples, in comparison with non-infected ones. Sample variability and the presence of potential biological outliers are verified using the molecular degree of perturbation approach in step 4. Finally, the functional analysis in step 5 reveals the pathways associated with the disease phenotype. The presented pipeline aims to support researchers through the RNA-seq data analysis from host-pathogen interaction studies and drive future in vitro or in vivo experiments, that are essential to understand the molecular mechanism of infections. (AU)

• FAPESP's process: 18/14933-2 - Integrative biology applied to human health
• Grantee: Helder Takashi Imoto Nakaya
• Support Opportunities: Research Grants - Young Investigators Grants - Phase 2
• FAPESP's process: 13/08216-2 - CRID - Center for Research in Inflammatory Diseases
• Grantee: Fernando de Queiroz Cunha
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 19/13880-5 - Leishmaniasis in Latin America: an advanced perspective on immunopatogenetic factors of cutaneous and visceral infection.
• Grantee: André Nicolau Aquime Gonçalves
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 19/16418-0 - Comprehensive meta-analysis of gene networks associated to human viral infections
• Grantee: Vanessa Escolano Maso
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 19/27146-1 - Integration of transcriptome data with clinical and immunological data
• Grantee: Amanda Pereira Vasconcelos
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 12/19278-6 - Systems biology of long non-coding RNAs
• Grantee: Helder Takashi Imoto Nakaya
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 20/05284-0 - Interaction of microorganisms and human hosts associated with the clinical outcome of patients with infectious diseases
• Grantee: Ícaro Maia Santos de Castro
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 17/50137-3 - Long noncoding RNA interplay with the host microbiome may determine mucosal influenza vaccine immunogenicity
• Grantee: Helder Takashi Imoto Nakaya
• Support Opportunities: Regular Research Grants
• FAPESP's process: 18/21934-5 - Network statistics: theory, methods, and applications
• Grantee: André Fujita
• Support Opportunities: Research Projects - Thematic Grants