Dual RNA-seq

Partial results are shown below:

Sequencing quality distribution

A/T/G/C Distribution

IGV Browser Interface

Correlation Analysis Between Samples

PCA Score Plot

Venn Diagram

Volcano Plot

Statistics Results of GO Annotation

KEGG Classification

1. Are reference genomes required for all species undergoing Dual RNA-seq?

Ideally, the availability of reference genomes for both interacting species would facilitate more accurate results. However, literature also documents procedures where only a single species has a reference genome at disposal. The analysis workflow in such cases entails initially mapping the sequencing data to the species with a reference genome. The transcriptomic data, post exclusion of mapping data, can be assayed for the other species' information through mapping with a close relative or a de novo assembly, facilitating subsequent analyses. Specifically, for the prokaryotic segment in an interacting sample, the presence of a reference genome is imperative. However, in its absence, bacterial 'pan-genome' profiling can be implemented.

2. What are the advantages of interacting transcriptomes over ordinary transcriptomes?

Dual RNA-seq is a specialized sequencing technique designed to probe interspecies interactions, generally encompassing symbiotic, parasitic, competitive, and predatory relationships. Traditional transcriptomics necessitate the separation of these interacting species, an approach which, albeit useful, can lead to loss of pertinent information and introduce potential artifacts related to the segregation process. In stark contrast is the dual RNA-seq approach, which enables simultaneous investigations of two or multiplex interacting species without the need for their partitioning. Consequently, this circumvents the possible detrimental influence incurred due to species separation. Moreover, this methodology is cost-effective, necessitating the construction of only one transcriptome library, which enables concurrent analysis of all included species.

3. What are the different types of RNA-seq?

Furthermore, depending on the research objectives and peculiarities of the sample, RNA-seq can be subdivided into several types such as: Total RNA-seq, mRNA-seq, miRNA-seq, Long Non-coding RNA-seq, Full-Length Transcriptome Sequencing, Circular RNA-seq, and Whole Exome RNA-seq.

Dual RNA-Seq Uncovers Metabolic Amino Acids Dependency of the Intracellular Bacterium Piscirickettsia salmonis Infecting Atlantic Salmon

Journal: Frontiers in microbiology
Impact factor: 6.064
Published: 27 November 2018

Abstract

High-throughput sequencing technologies, applied to transcriptomic research (RNA-Seq), have opened up the possibility of comprehending complex molecular reactions within different biological contexts. Recently, the synchronous sequencing of a pathogen's and host's transcriptomes during infection - known as dual RNA-seq - has emerged as a promising method to unveil the intricacies of host-pathogen interactions. In this particular study, through dual rRNA depletion and RNA sequencing scheme, researchers simultaneously analyzed the transcriptomes of the intracellular bacterium Piscirickettsia in salmon and its host species, Salmo salar, throughout the course of the infection.

Methods

Results

Exploring Host and Pathogen Transcriptome During Pathogenesis

Dual RNA-Seq analysis delineated the regulation of the transcriptomes of P. salmonis and S. salar during the course of infection. Regarding the S. salar transcriptome, 771 and 829 genes were differentially expressed in the spleen at 7 and 14 dpi respectively (Fig 1B). Concurrently, 412 and 467 genes were differentially regulated at these same time points in the head kidney. Genes specifically modulated in the head kidney included those encoding MATE efflux family protein family protein, DNA repair protein RecN, chaperone protein HtpG and various membrane proteins (Fig 2). Among the shared genes, different protein phosphatase 1, ribosomal proteins, molecular chaperones and Asn/Gln transamidosome subunits were identified (Fig 2).

FIGURE 1. (A) Global transcriptome analysis of Piscirickettsia salmonis (blue) and S. salar (red) at 3, 7, and 14 days post-infection (dpi) in spleen and head kidney tissues. Expression values were estimated as transcripts per million reads (TPM) and Log2 transformed for heat map clustering. (B) Differentially expressed genes in spleen and head kidney at 7 and 14 dpi using as control the expression values at 3 dpi. Three days post-infection were used as baseline gene expression since no bacterial reads would be present in any type of control group.

FIGURE 2. Venn diagram showing the number of exclusive and shared genes in the transcriptome of P. salmonis during the infection in spleen (blue) and head kidney (red) tissues of Atlantic salar. The heat maps show a subset of exclusive and shared genes expressed by P. salmonis in both tissues.

Amino Acid Metabolism: A Common Response Between P. salmonis and S. salar

The GO enrichment analysis indicated that a large proportion of the differentially expressed genes in P. salmonis were associated with protein and nitrogen compound metabolic processes. Enrichment of genes related to protein metabolism was also discovered via KEGG annotation. However, despite amino acid metabolism not being the major transcriptomic response within the host, both P. salmonis and S. salar demonstrated numerous genes connected to the biological synthesis and degradation of amino acids.

FIGURE 3. Gene ontology annotation (A) and KEGG pathway annotation (B) of the differentially expressed genes in Salmo salar and P. salmonis during the infection.

Conclusion

Beyond the standard host immune responses and the virulence factors of pathogens, both bacteria and host exhibit a profusion of genes associated with metabolic processes, in particular, those relating to amino acid metabolism. Our findings point to a dependency of P. salmonis on the amino acid metabolism of S. salar, which could suggest a novel pathogenesis mechanism grounded in the capacity to absorb nutrients from the host. In broad terms, dual transcriptome sequencing permits a varied perspective on the interaction between host and pathogen, transcending beyond the biological processes intertwined with immunity.

Reference:

1. Valenzuela-Miranda D, Gallardo-Escárate C. Dual RNA-Seq uncovers metabolic amino acids dependency of the intracellular bacterium Piscirickettsia salmonis infecting Atlantic salmon. Frontiers in microbiology, 2018, 9: 418416.