Check list

Once the pipeline has ended successfully, please check the multiqc_report.html file to see if there is any problem.

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Here is non exhaustive list of sanity checks

• General statistics : percentage of aligned reads according to sample name
• FeatureCounts : Assigned vs Unassigned
• Alignment scores : According to mapper provided data Mapped vs Unmapped
• Bowtie 1 : rRNA mapped data should be low less than 10-20 % ???
• Cutadapt : Trimmed sequence length should be less than 30pb
• FastQC : Reads quality
• Sequence Quality Histograms : Curve has to be flat
• Per Sequence GC Content : Gives an insight of the GC composition for a given experiment. Usually around 50% of GC
• Per Base N Content: Gives an insight of the undetermined bases for a given experiment. Hopefully around 0% of N

Check RNAdiff report

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• Table 1 : Check file IDs and labels
• Table 3 to X : Summary of the count tables
• Figure 1 : Check if mapped reads are similar or not
• Figure 2 : Check percentage of null counts (the dotted line) that will be set to NA (log fold change and p-value) in the result table
• Figure 3 : Check the denity of raw counts. They must follow the same pattern
• Figure 4 : Check the percentage of reads associated with the gene with the higher count. It should/could be the same in all replicate for one condition. Gives an insight if the highest count gene for each condition.
• Figure 5 : The sample with similar conditions should/could cluster together
• Figure 6 : First 2 component of the PCA : PC1 = X percentage of the variance. Usually what is expected is PC1 80% PC2 20% for a clear differential expression
• Figure 7 : Check if Boxplot comparison that illustrate normalisation is OK, They should be aligned after normalisation according to the median.
• Figure 8 :
• Figure 9 : Highest histogram bar should be around 0
• Figure 10 and 11 : MAplot and volcano plot showing diffrentialy expressed genes. The 0 axis cut the graph in two parts : Up regulated and down regulated

Results

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• Create a folder to report results
• Copy in this folder : the pdf report from Deseq2 the tables end figures folder fron Deseq2 the multiqc html report Any other asked files