featureCounts
- In your history
HISAT2
orSTAR
- Select the
featureCounts
tool with the following parameters to count your reads:Alignment file
: select multiple datasets button and shift-click the 7 bam files you have generatedSpecify strand information
: UnstrandedGene annotation file
: in your historyGene annotation file
: Drosophila_melanogaster.BDGP6.95.gtf
FASTA/Q file
: Gene-ID "\t" read-count (MultiQC/DESeq2/edgeR/limma-voom compatible)Create gene-length file
: Yes- In
Options for paired-end reads
:Count fragments instead of reads
: Enabled; fragments (or templates) will be counted instead of reads
- In
Advanced options
:GFF feature type filter
: exonGFF gene identifier
: gene_idAllow read to contribute to multiple features
: NoCount multi-mapping reads/fragments
: Disabled; multi-mapping reads are excluded (default)Minimum mapping quality per read
: 10
- Leave other settings as defaults
Execute
You need now to rename you datasets to facilitate your downstream analysis.
Be quiet and focus ! No hurry, this is an important task in the analysis.
-
Search and select datasets with
featurecounts
(as we did before for renaming datasets) -
Click on the info icon of
featureCounts on xxx: Counts
-
Copy the name of the dataset
Alignment file
in the tool parameters table (for instance,GSM461176_untreat_single.bam
) - Now click on the pencil icon of the same dataset
- Paste your text in the
Name
field of the dataset - Edit your text by replacing
bam
byCounts
(e.g. GSM461176_untreat_single.Counts) - repeat ad lib for all counts files generated by featureCounts
MultiQC
We have now generated (1) Bam alignments and (2) Counts files with feature counts, and we have carefully and courageously edited the names of generated datasets. We are going to be rewarded for this effort in the next steps !
- In your history
HISAT2
orSTAR
- Select the
MultiQC
tool with the following parameters:1: Results
/Which tool was used generate logs?
: STARor
HISAT2 (depending on your analysis track)STAR or HISAT output
: shift-click select all files with the extension .log- Click on the
+ Insert Result
button 2: Results
/Which tool was used generate logs?
: featureCountsOutput of FeatureCounts
: shift-click select all files with the extension : Summary
Execute
Examine the results and