--- title: "Transcript-Level Analysis" canonical: "https://help.biobam.com/space/OED0324/3525083289/Transcript-Level%20Analysis" format: markdown --- # Introduction Transcriptome Analysis of *Monilinia fructigena.* ### Dataset Description Transcriptomes of *Monilinia fructicola*, ***Monilinia laxa****,* and *Monilinia fructigena*, the causal agents of brown rot of stone and pome fruits. For this tutorial, only the data of *Monilinia laxa* is used. This dataset comprises paired-end reads that were corresponding to mycelium grown in the dark for 4 days, mycelium grown in the dark for 2 days, and then exposed to light for 2 days, as well as in germinating conidia (2 replicates per each condition). - Organism: *[Monilinia laxa](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=61186&lvl=3&lin=f&keep=1&srchmode=1&unlock)* - Instrument: Illumina HiScanSQ - Layout: Paired-end ### Publication [De Miccolis Angelini RM, Abate D, Rotolo C, Gerin D, Pollastro S, Faretra F. De novo assembly and comparative transcriptome analysis of Monilinia fructicola, Monilinia laxa and Monilinia fructigena, the causal agents of brown rot on stone fruits. BMC Genomics. 2018 Jun 5;19(1):436. doi: 10.1186/s12864-018-4817-4. PMID: 29866047; PMCID: PMC5987419.](https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4817-4)*[Monilinia fructicola](https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4817-4)*[, ](https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4817-4)*[Monilinia laxa](https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4817-4)*[ and ](https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4817-4)*[Monilinia fructigena](https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4817-4)*[, the causal agents of brown rot on stone fruits.](https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4817-4)
Abstract Brown rots are important fungal diseases of stone and pome fruits. They are caused by several *Monilinia* species but *M. fructicola*, *M. laxa,* and *M. fructigena* are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled, and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per species for comparative transcriptome analyses.
### Original Data - NCBI BioProject: [PRJNA419302](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA419302) - SRA Experiments: [SRR6312174, SRR6312175, SRR6312181, SRR6312182, SRR6312187, and SRR6312190](https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP125382&o=acc_s%3Aa). - NCBI Nucleotide: [TSA: ](https://www.ncbi.nlm.nih.gov/nuccore/GGAL00000000.1)*[Monilinia laxa](https://www.ncbi.nlm.nih.gov/nuccore/GGAL00000000.1)*[, transcriptome shotgun assembly.](https://www.ncbi.nlm.nih.gov/nuccore/GGAL00000000.1) # Bioinformatic Analysis ## 1- Expression Quantification ### Application [Create Count Table, Transcript-level Quantification](https://docs.omicsbox.biobam.com/latest/Transcript-level-Quantification/) (Transcriptomics). ### Input - [Sequencing data in FASTQ format (SRR6312174, SRR6312175, SRR6312181, SRR6312182, SRR6312187, and SRR6312190). ](https://drive.google.com/drive/u/1/folders/1v7H0B3TmUOhcHqLb_B-gb03MGy1ThIi_) - [CDS sequences from transcriptome assembly](https://drive.google.com/file/d/11IB6kMwkaACU_wM9szvr1Z9dCZbxK8id/view?usp=sharing) (from the [https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization](https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization) tutorial, [4- Predict Coding Regions](https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization#4--Predict-Coding-Regions) step). ### Parameters - Input Reads: [Paired-end] SRR6312174, SRR6312175, SRR6312181, SRR6312182, SRR6312187, and SRR6312190 FASTQ files - Upstream Files Pattern: _1 - Downstream Files Pattern: _2 - Input FASTA: cds.box - Gene-level Estimations: false - Estimate RSPD: true - Append Poly(A) Tails: false - Strand Specificity: Non-Strand Specific - Provide Fragment Length Distribution: false - Generate Alignment Files: false ### Execution Time 30-40 minutes. ### Output - [counts.box → Project containing expression values for each gene and each sample.](https://drive.google.com/file/d/1JbNCrbgzLPwo5YROfG4p9-z15Y0mKowv/view?usp=sharing) ## 2- Differential Expression Analysis ### Application [Pairwise Differential Expression Analysis](https://docs.omicsbox.biobam.com/latest/Pairwise-Differential-Expression-Analysis/) (Transcriptomics). ### Input - [Count Table](https://drive.google.com/file/d/1JbNCrbgzLPwo5YROfG4p9-z15Y0mKowv/view?usp=sharing) (from the [1- Expression Quantification](https://biobam.atlassian.net/wiki/spaces/OED0324/pages/3525083289/Transcript-Level+Analysis#1--Expression-Quantification) step). - [Experimental Design.](https://drive.google.com/file/d/13_ifsD5bVBclrRJeBQL5zSlb9ueKFdPA/view?usp=sharing) ### Parameters - CPM Filter: 0.0 - Samples reaching CPM Filter: 1 - Normalization Method: TMM (Trimmed mean of M values) - Design Type: Simple Design - Primary Experimental Factor: Condition - Primary Contrast Condition: dark_4_days - Primary Reference Condition: getminating_conidia - Select a Statistical Test: Exact Test - Robust: True ### Execution Time 5-10 minutes. ### Output - [dark_4_days_vs_germinating_counts.box → Project containing results of pairwise differential expression analysis.](https://drive.google.com/file/d/1iJG0Og9Mqfc8Xlw2bBkY3_DW17V4qMYS/view?usp=sharing) ## 3- Enrichment (GSEA) ### Application [Gene Set Enrichment Analysis](https://docs.omicsbox.biobam.com/latest/Pairwise-Differential-Expression-Analysis/#gene-set-enrichment-analysis). ### Input - [Differential Expression Result](https://drive.google.com/file/d/1iJG0Og9Mqfc8Xlw2bBkY3_DW17V4qMYS/view?usp=sharing)s (from the [2- Differential Expression Analysis](https://biobam.atlassian.net/wiki/spaces/OED0324/pages/3525083289/Transcript-Level+Analysis#2--Differential-Expression-Analysis) step). - *[M. laxa ](https://drive.google.com/file/d/1LBBweuwUdyrqlhKrZu2jy_Hsu57YSNhG/view?usp=sharing)*[functional annotation](https://drive.google.com/file/d/1LBBweuwUdyrqlhKrZu2jy_Hsu57YSNhG/view?usp=sharing) (from the [https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization](https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization) tutorial, [5- Functional Annotation](https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization#5--Functional-Annotation) step). ### Parameters - Reference Annotation: protein.box - Number of Permutations: 1000 - Enrichment Statistic: Classic - Number of Detailed Results: 50 - Detailed Results for All GOs: false - GO Categories: biological_process,molecular_function,cellular_component - Gene Sets Max Size: 500 - Gene Sets Min Size: 15 - Do Not Filter: false - Filter Mode: FDR - Filter Value: 0.25 ### Execution Time 5 minutes. ### Output - [gsea_results.box: Project containing results of the functional enrichment analysis.](https://drive.google.com/file/d/12-xTZ972DrExZ1xlKb22FDcCOHc8OL_s/view?usp=sharing) ## 4- Enrichment (Fisher’s Exact Test) ### Application [Fisher’s Exact Test](https://docs.omicsbox.biobam.com/latest/Pairwise-Differential-Expression-Analysis/#fishers-exact-test). ### Input - [Differential Expression Result](https://drive.google.com/file/d/1iJG0Og9Mqfc8Xlw2bBkY3_DW17V4qMYS/view?usp=sharing)s (from the [2- Differential Expression Analysis](https://biobam.atlassian.net/wiki/spaces/OED0324/pages/3525083289/Transcript-Level+Analysis#2--Differential-Expression-Analysis) step). - *[M. laxa ](https://drive.google.com/file/d/1LBBweuwUdyrqlhKrZu2jy_Hsu57YSNhG/view?usp=sharing)*[functional annotation](https://drive.google.com/file/d/1JKrd_vCC8NPo1pHlboEwUNNiGXPxYZsC/view?usp=sharing) (from the [https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization](https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization) tutorial, [5- Functional Annotation](https://help.biobam.com/space/OED0324/3525083363/De+Novo+Transcriptome+Characterization#5--Functional-Annotation) step). ### Parameters - Test-Set Genes: Up-regulated genes - Reference Annotation: protein.annot - 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 5 minutes. ### Output - [go_ids_fisher_result.box: Project containing results of the functional enrichment analysis.](https://drive.google.com/file/d/1Ejs3QzQO-BJiisB2pOy6l1coGrOEeurq/view?usp=sharing) ## Workflow ![image](media://ac4a0939-2dcb-46a9-a8b7-509bffa9feb5)