galaxyproject / galaxyproject/training-material

Reference-based RNA-Seq tutorial - places it's out of sync with usegalaxy.org

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CoFest Hacktoberfest help-wanted newcomer-friendly paper-cut transcriptomics
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Description

I went through the tutorial, following the steps on usegalaxy.org. Here are places where the instructions or results in the tutorial differed from what I saw on Main. I executed every step where it suggests running a tool on a dataset, including ones mentioned inside collapsible sections like "Questions", "Solution", or "Tip".

## [Data upload](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#data-upload)
### Hands-on: Data upload > 2. Import the FASTQ file pairs > Comment
The [Zenodo link](https://zenodo.org/record/1185122) to the subset FASTQ files doesn't contain them. There aren't any FASTQ files smaller than 1.4GB.

## [Mapping](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#mapping-1)
### Hands-on: Spliced mapping > 2. **RNA STAR**
It says to use the reference `Fly (Drosophila Melanogaster): dm6 Full` but currently on usegalaxy.org, the only `dm6` is `D. melanogaster Aug. 2014 (BDGP Release 6 + ISO1 MT/dm6) (dm6)`

## [Inspection of the mapping results](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#inspection-of-the-mapping-results)
### Further check for the quality of the data > Number of reads mapped to each chromosome > Hands-on: Check the number of reads mapped to each chromosome > 2. **MultiQC**
The instructions say to select `idxstats` for "Which tool was used generate logs?", but that parameter should be "Samtools" and the "Type of Samtools output?" should be `idxstats` (I assume)

## [Counting reads per genes](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#counting-reads-per-genes)
### Hands-on: Counting the number of reads per annotated gene > 1. **featureCounts** > In "*Advanced options*"
"*Allow read to contribute to multiple features*" should be "*Allow reads to map to multiple features*" (I assume)
and
`No` should be `Disabled; reads that align to multiple features or overlapping features are excluded` (I assume)

But then the results look different from what's shown in the "Solution" box. There are two additional categories included:
- `Unassigned_Unmapped`
- `Unassigned_MappingQuality`

### Question > Solution
It would be good to specify *which* output of featureCounts to sort (the `Counts` one)

## [Extraction and annotation of differentially expressed genes](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#extraction-and-annotation-of-differentially-expressed-genes)
### Hands-on: Add column names > 2. Concatenate datasets
It might be good to specify *which* **Concatenate datasets** tool to use. On usegalaxy.org there's "**Concatenate datasets** tail-to-head (cat)" and "**Concatenate datasets** tail-to-head".
Same with the **Cut** steps later on:
- [Visualization of the normalized counts](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#extraction-and-annotation-of-differentially-expressed-genes) > Hands-on: Extract the normalized counts of the most differentially expressed genes
- [Gene Ontology analysis](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#gene-ontology-analysis) > Hands-on: Prepare the datasets for goseq
- [KEGG pathways analysis](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#kegg-pathways-analysis) > Hands-on: Perform KEGG enrichment analysis

## [Visualization of the normalized counts](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#visualization-of-the-normalized-counts)
The heatmap I got is a bit different: [Galaxy22-[heatmap2_on_data_21].pdf](https://github.com/galaxyproject/training-material/files/5978463/Galaxy22-.heatmap2_on_data_21.pdf)

## [Computation and visualization of the Z-score](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#computation-and-visualization-of-the-z-score)
The heatmap I got is slightly different: [Galaxy25-[heatmap2_on_data_24].pdf](https://github.com/galaxyproject/training-material/files/5978468/Galaxy25-.heatmap2_on_data_24.pdf)

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