A representative analysis can be performed using the RNA-seq data SRA accession SRR319229 that was the Wold dataset that was used in the original analysis of bias.

This can be done using a prealigned bam file that can be downloaded. Alternatively the bam file can be created by downloading and aligning the raw read data as follows:

The raw data can be downloaded. Its recommended that this is done by installing the SRA toolkit using the command:

fastq-dump SRR319229

This can then be aligned to a reference genome using hisat2 which can be downloaded and then aligned using the following command:

hisat2 –p 10 -x grcm38_tran/genome_tran -U SRR3192229.fastq -S SRR3192229.sam
samtools view -b SRR3192229.sam > SRR3192229.bam

where genome_tran is the root name of the reference genome that can be downloaded 

After the bam file has either been downloaded or created, normalisation of these data can then be performed by LiBiNorm using the following command:

LiBiNorm count -p 3 -f -s no -u wold -c counts.txt SRR3192229.bam Mus_musculus.GRCm38.80.gtf

This specifies that all the models are evaluated but that model BD is nevertheless used to correct for the bias. Auxiliary output files all have the prefix wold and the expression data is output to counts.txt. Three threads are used.

This requires a reference annotation that can be downloaded and uncompressed. This will produce these described output data files.

The graphical representations can be produced using the command:

Rscript LiBiNormPlot.R wold