Transcript-Level Analysis
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
Instrument: Illumina HiScanSQ
Layout: Paired-end
Publication
Original Data
NCBI BioProject: PRJNA419302
SRA Experiments: SRR6312174, SRR6312175, SRR6312181, SRR6312182, SRR6312187, and SRR6312190.
NCBI Nucleotide: TSA: Monilinia laxa, transcriptome shotgun assembly.
Bioinformatic Analysis
1- Expression Quantification
Application
Create Count Table, Transcript-level Quantification (Transcriptomics).
Input
CDS sequences from transcriptome assembly (from the https://biobam.atlassian.net/wiki/pages/resumedraft.action?draftId=2377941020 tutorial, 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
2- Differential Expression Analysis
Application
Pairwise Differential Expression Analysis (Transcriptomics).
Input
Count Table (from the 1- Expression Quantification step).
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
3- Enrichment (GSEA)
Application
Gene Set Enrichment Analysis (Functional Analysis).
Input
Differential Expression Results (from the 2- Differential Expression Analysis step).
M. laxa functional annotation (from the https://biobam.atlassian.net/wiki/pages/resumedraft.action?draftId=2377941020 tutorial, 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
4- Enrichment (Fisher’s Exact Test)
Application
Fisher’s Exact Test (Functional Analysis).
Input
Differential Expression Results (from the 2- Differential Expression Analysis step).
M. laxa functional annotation (from the https://biobam.atlassian.net/wiki/pages/resumedraft.action?draftId=2377941020 tutorial, 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.