Mutant Transcript is higher than the WT. What may be the reason?

Question

[WUSCHEL]

I used "Kallisto" pseudoaligning methods and have done my RNASeq analysis.

One of my T-DNA knockout plants shows higher KO gene expression compared to the WT.

These are published lines and I have already genotyped them by PCR and conformed they are T-DNA mutants.

first I thought this is because of pesedomapping and with the feedback from BioStars community, later I generated .BMA & .bai files and visually examined the reads on IGV browser. bamCoverage is high in the mutants.

What could be the reason? Are there any other Bioinformatics tools to check this?

[ATpoint]

What do we see there? There aren't any scales. All I see is what looks like some low-coverage noise in the red area. Please zoom in. Also, coverage tracns directly from BAM files are not normalised for read depth, therefore you cannot compare them. Use normalized tracks, at least for read depth, e.g. bamCoverage from deeptools. What kind of KO is this (how was it made)? Is this a point mutation that renders the protein non-functional or was an exon cut out?

[Vitis]

Comparing expression levels from IGV views is not very informative. You need to at least normalize against library size to compare expression levels of a genes across samples.

[WUSCHEL]

Thank you @ATpoint and @Vitis.

These are T-DNA mutants.

May I ask if there is any protocol I can find to do what both of you have suggested. I am not a Bioinformatician tbh.

[Vitis]

One simple way to try this is: for each sample, count total number of reads mapped within the gene boundaries, divide by the number of total reads, then multiply 1 million. This gives you the reads/million for this gene in this sample, so you can compare this value across samples. Basically, for each sample (mutant or WT),

normalized_counts = gene_read_counts/sample_total_reads*1,000,000

The next step is to find a collaborator who is familiar with mRNA-Seq analysis and do a proper differential expression analysis between mutant and WT with replicates.