--- title: "Gene-Level Analysis" canonical: "https://help.biobam.com/space/OBD/2378039311/Gene-Level%20Analysis" format: markdown --- # Introduction Transcriptomes of FA1090, FA1090cpxA::kan, FA1090cpxR::erm mutant strains of *Neisseria gonorrhoeae*. ### Dataset Description RNA of *Neisseria gonorrhoeae* wildtype, cpxA, and cpxR mutants were collected at late log phase of growth, in quadruplicate (12 libraries in total). - Organism: *[Neisseria gonorrhoeae](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=485)**.* - Instrument: Illumina NextSeq 500. - Layout: Single-end. ### Publication [Gangaiah D, Raterman EL, Wu H, Fortney KR, Gao H, Liu Y, Jerse AE, Spinola SM. Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection in a Murine Model of Lower Genital Tract Infection. Infect Immun. 2017 Aug 18;85(9):e00307-17. doi: 10.1128/IAI.00307-17. PMID: 28652307; PMCID: PMC5563589.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563589/)
Abstract During infection, *Neisseria gonorrhoeae* senses and responds to stress; such responses may be modulated by MisRS (NGO0177 and NGO0176), a two-component system that is a homolog of CpxRA. In *Escherichia coli*, CpxRA senses and responds to envelop stress; CpxA is a sensor kinase/phosphatase for CpxR, a response regulator. When a *cpxA* mutant is grown in medium containing glucose, CpxR is phosphorylated by acetyl phosphate but cannot be dephosphorylated, resulting in constitutive activation. Kandler and coworkers (J. L. Kandler, C. L. Holley, J. L. Reimche, V. Dhulipala, J. T. Balthazar, A. Muszyński, R. W. Carlson, and W. M. Shafer, Antimicrob Agents Chemother 60:4690–4700, 2016, [https://doi.org/10.1128/AAC.00823-16](https://doi.org/10.1128/AAC.00823-16) ) showed that MisR (CpxR) is required for the maintenance of membrane integrity and resistance to antimicrobial peptides, suggesting a role in gonococcal survival *in vivo*. Here, we evaluated the contributions of MisR and MisS (CpxA) to gonococcal infection in a murine model of cervicovaginal colonization and identified MisR-regulated genes using RNA sequencing (RNA-Seq). The deletion of *misR* or *misS* severely reduced the capacity of *N. gonorrhoeae* to colonize mice or maintain infection over a 7-day period and reduced microbial fitness after exposure to heat shock. Compared to the wild type (WT), the inactivation of *misR* identified 157 differentially regulated genes, most of which encoded putative envelope proteins. The inactivation of *misS* identified 17 differentially regulated genes compared to the WT and 139 differentially regulated genes compared to the *misR* mutant, 111 of which overlapped those differentially expressed in the comparison of the WT versus the *misR* mutant. These data indicate that an intact MisRS system is required for gonococcal infection of mice. Provided the MisR is constitutively phosphorylated in the *misS* mutant, the data suggest that controlled but not constitutive activation is required for gonococcal infection in mice.
### Original Data - NCBI BioProject: [PRJNA325641](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA325641). - SRA Experiments: [SRR3666079-SRR3666090](https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP076546&o=acc_s%3Aa). - GEO Dataset: [GSE83341](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE83341). - Reference Genome: [Neisseria gonorrhoeae FA 1090 (GCA_000006845)](https://bacteria.ensembl.org/Neisseria_gonorrhoeae_fa_1090_gca_000006845/Info/Index/). # Bioinformatic Analysis ## 1- RNA-Seq Alignment ### Application [RNA-Seq Alignments](https://biobam.atlassian.net/wiki/spaces/OBD/pages/618856449), [BWA ](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2519302295)(Transcriptomics). ### Input - [Sequencing data in FASTQ format (SRR3666079-SRR3666090)](https://drive.google.com/drive/u/1/folders/1yP1PNpWoTKbCmrr3c0d8ILhVD5_LqwQH). - *[N. gonorrhoeae](https://drive.google.com/file/d/1KMbDrtchf9WQNGh-2XuRfwj-MHdUX1i5/view?usp=sharing)*[ reference genome](https://drive.google.com/file/d/1KMbDrtchf9WQNGh-2XuRfwj-MHdUX1i5/view?usp=sharing). ### Parameters ### Execution Time 10-15 minutes. ### Output - [BAM files: 12 BAM files containing alignments from the RNA-Seq Alignment task](https://drive.google.com/drive/u/1/folders/1HP2nDLxBz4C2aQl-IWfVNDlvTiIvewza). - [read_alignment_bwa.box: Report about the RNA-Seq Alignment results](https://drive.google.com/file/d/1IqoDGXNluefU-2V5UbKQkg47x7j91wTR/view?usp=sharing). - [alignments_per_category.box: Bar plot about the RNA-Seq Alignments results](https://drive.google.com/file/d/1dKmPhJ7QlsQK6UfOsL2C7nApZ-PzVhae/view?usp=sharing). - [relative_alignments_per_category.box: Bar plot about the RNA-Seq Alignments results (in percentages)](https://drive.google.com/file/d/1taXwMusHSJ-PVua6QXa6_7xWTlGQuye-/view?usp=sharing). ## 2- Expression Quantification ### Application [Create Count Table](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2522644495), [Gene-level Quantification](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598114455) (Transcriptomics). ### Input - [RNA-Seq Alignments in BAM format](https://drive.google.com/drive/u/1/folders/1HP2nDLxBz4C2aQl-IWfVNDlvTiIvewza) (from the [1- RNA-Seq Alignment](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2378039311/Gene-Level+Analysis#1--RNA-Seq-Alignment) step). - *[N. gonorrhoeae](https://drive.google.com/file/d/1KDl2Pvxv9zFB6bDEjZzM0Ahdeqozt8NA/view?usp=sharing)*[ reference annotation in GTF format](https://drive.google.com/file/d/1KDl2Pvxv9zFB6bDEjZzM0Ahdeqozt8NA/view?usp=sharing). ### Parameters - Input BAMs: SRR3666079-SRR3666090 BAM files - Input GFF/GTF: /00_reference/Neisseria_gonorrhoeae_fa_1090.ASM684v1.47.gtf - Quantification Level: gene - Name/Group By: gene_id: - Strand Specificity: Non Strand Specific - Overlap Mode: Union - Minimum Mapping Quality: 10 ### Execution Time 5-10 minutes. ### Output: - [counts.box: Project containing expression values for each gene and each sample](https://drive.google.com/file/d/1BgVOijfm8D8eQ3HLey1MEOpo4MW7vwRj/view?usp=sharing). ## 3- Differential Expression Analysis ### Application [Pairwise Differential Expression Analysis](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598081602) (Transcriptomics). ### Input - [Count Table](https://drive.google.com/file/d/1BgVOijfm8D8eQ3HLey1MEOpo4MW7vwRj/view?usp=sharing) (from the [2- Expression Quantification](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2378039311/Gene-Level+Analysis#2--Expression-Quantification) step). - [Experimental Design](https://drive.google.com/file/d/1Jwil11PF5-wem54nYn9n9sy_LDBvkxli/view?usp=sharing). ### Parameters - CPM Filter: 0.0 - Samples reaching CPM Filter: 1 - Normalization Method: TMM (Trimmed mean of M values) - Experimental Design File: experimental_design.txt - Design Type: Simple Design - Primary Experimental Factor: Strain - Primary Contrast Condition: FA1090cpxR - Primary Reference Condition: FA1090 - Select a Statistical Test: Exact Test - Robust: true ### Execution Time 5-10 minutes. ### Output - [fa1090cpxr_vs_fa1090_counts.box: Project containing results of the pairwise differential expression analysis](https://drive.google.com/file/d/1uvKnfUySsYD0J_XADiFiLXC0HReNx4pH/view?usp=sharing). ## 4- Enrichment (GSEA) ### Application [Gene Set Enrichment Analysis](https://biobam.atlassian.net/wiki/spaces/OBD/pages/597917886) (Functional Analysis). ### Input - [Differential Expression Results](https://drive.google.com/file/d/1uvKnfUySsYD0J_XADiFiLXC0HReNx4pH/view?usp=sharing) (from the [3- Differential Expression Analysis](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2378039311/Gene-Level+Analysis#3--Differential-Expression-Analysis) step). - *[N. gonorrhoeae](https://drive.google.com/file/d/1Hk1h4J7Zo9IRs13rfw2vq3IZPsHwxFYa/view?usp=sharing)*[ functional annotation](https://drive.google.com/file/d/1Hk1h4J7Zo9IRs13rfw2vq3IZPsHwxFYa/view?usp=sharing). ### Parameters - Reference Annotation: /00_reference/neisseria_gonorrhoeae_functions.box - FDR Filter for Ranked List: 1.0 - Number of Permutations: 1000 - Enrichment Statistic: Classic - Detailed Results for All GOs: false - Number of Detailed Results: 50 - GO Categories: biological_process,molecular_function,cellular_component - Gene Sets Max Size: 500 - Gene Sets Min Size: 15 - Filter Mode: FDR - Filter Value: 0.25 ### Execution Time 1-2 minutes. ### Output - [gsea_results.box: Project containing results of the functional enrichment analysis](https://drive.google.com/file/d/1G1z2QyPfXMWP5sEK97-ctCAEXzllZb9Y/view?usp=sharing). ## 5- Enrichment (Fisher’s Exact Test) ### Application [Fisher’s Exact Test](https://biobam.atlassian.net/wiki/spaces/OBD/pages/597983330) (Functional Analysis). ### Input - [Differential Expression Results](https://drive.google.com/file/d/1uvKnfUySsYD0J_XADiFiLXC0HReNx4pH/view?usp=sharing) (from the [3- Differential Expression Analysis](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2378039311/Gene-Level+Analysis#3--Differential-Expression-Analysis) step). - *[N. gonorrhoeae](https://drive.google.com/file/d/1Orn4ylB-97kJGO6tp9B9taqRTXBg_pVK/view?usp=sharing)*[ functional annotation](https://drive.google.com/file/d/1Orn4ylB-97kJGO6tp9B9taqRTXBg_pVK/view?usp=sharing). ### Parameters - Reference Annotation: neisseria_gonorrhoeae_functions.annot - Test-Set Genes: Up-regulated genes - Do Not Filter: false - Filter Value: 0.05 - Two Tailed: false - Remove Double IDs: false - Filter Mode: FDR - Annotations: GO IDs - GO Categories: biological_process,molecular_function,cellular_component ### Execution Time 1-2 minutes. ### Output: - [go_ids_fisher_result.box: Project containing results of the functional enrichment analysis](https://drive.google.com/file/d/1_YOxYtneUa7qTzdHaR5wJxPMtDbYO-jd/view?usp=sharing). ## Workflow Gene-level analysis workflow. ![image](media://c77d5012-238a-46d0-8f66-a3f289922900)