Sum of PSI values in an LSV does not equal 1

[Conall Moore]

Hi Biociphers,

Thank you for the amazing tool!

I have previously presented data from MAJIQ PSI by generating stacked bar charts for an entire LSV with PSI values on the y-axis, conditions on the x-axis, and each stack is a splice junction. That way, the sum of PSI values for all junctions in the LSV is approximately 1.0 for each condition. I've attached a diagram for clarity. 

Using the same MAJIQ build, I recently ran this data through HET and for some conditions, the sum of all junction PSIs was quite a bit lower than expected (as low as 0.86 in some cases). Do you have any idea why this could be the case? I could normalise the PSI values for each condition, but I'm just curious as to what could be the cause here.

I've outlined my commands below.

Any help would be greatly appreciated!

Conall

_______________

Trimming:

~/TrimGalore-0.6.10/trim_galore --paired --phred33 --fastqc --gzip -j 8 fastq.gz

Alignment:

STAR --runMode alignReads --genomeDir genome_directory/ --sjdbOverhang 99 --readFilesCommand zcat --outSAMtype BAM SortedByCoordinate --readFilesIn $R1 $R2 --outFileNamePrefix path/${SAMPLE}_ --runThreadN 8

Build:

config file:

[info]

readlen=100

bamdirs=sorted_by_coordinate_bams/

genome=hg38

genome_path=genome.fa

[experiments]

group1=g1_Aligned.sortedByCoord.out,g2_Aligned.sortedByCoord.out,g3_Aligned.sortedByCoord.out

group2=h1_Aligned.sortedByCoord.out,h2_Aligned.sortedByCoord.out,h3_Aligned.sortedByCoord.out

/home/user/majiq/bin/majiq --license majiq_license build genome.annotation.gff3 -o output -c config.ini -j 4 --minreads 10

PSI:

majiq --license majiq_license psi -o output -n group1 g1_Aligned.sortedByCoord.out.majiq g2_Aligned.sortedByCoord.out.majiq g3_Aligned.sortedByCoord.out.majiq 

majiq --license majiq_license psi -o output -n group2 h1_Aligned.sortedByCoord.out.majiq h2_Aligned.sortedByCoord.out.majiq h3_Aligned.sortedByCoord.out.majiq 

Heterogen:

majiq --license majiq_license heterogen -j 4 -o output -grp1 g1_Aligned.sortedByCoord.out.majiq g2_Aligned.sortedByCoord.out.majiq g3_Aligned.sortedByCoord.out.majiq  -grp2 h1_Aligned.sortedByCoord.out.majiq h2_Aligned.sortedByCoord.out.majiq h3_Aligned.sortedByCoord.out.majiq  -n group1 group2 --stats TNOM TTEST WILCOXON

Voila:

voila --license majiq_license tsv splicegraph.sql group1-group2.het.voila -f group1_group2_het_voila.tsv