STARsolo intergenic reads as same as antisense

[Liripo]

Hello developer:
I use STARsolo to analyze SPLit-seq data. My command is:

STAR \
    --runMode alignReads \
    --soloCellFilter EmptyDrops_CR \
    --runThreadN 16 \
    --genomeDir ${star_index} \
    --readFilesIn ${R1_fq} ${R2_fq} \
    --readFilesCommand zcat \
    --outSAMattributes NH HI nM AS CR UR CB UB GX GN sS sQ sM \
    --outSAMtype BAM SortedByCoordinate \
    --soloCBwhitelist ${barcode_list1} ${barcode_list2} ${barcode_list3} \
    --soloType CB_UMI_Complex \
    --soloCBposition 0_10_0_17 0_48_0_55 0_86_0_93 \
    --soloUMIposition 0_0_0_9 \
    --soloCBmatchWLtype EditDist_2 \
    --soloFeatures GeneFull_Ex50pAS \
    --soloMultiMappers EM \
    --soloCellReadStats Standard \
    --outSAMunmapped Within

I use --soloFeatures GeneFull_Ex50pAS, and use the output file Solo.out/GeneFull_Ex50pAS/CellReads.stats to calculate the ratio of intergenic regions and antisense genes.However I got exactly the same reads. I calculated it like this：

intergenic = genomeU + genomeM - exonic - intronic
antisense = exonicAS + intronicAS

Is it because the reads aligned to non-introns and non-exons are classified as antisense?
Thank you!

[alexdobin]

Hi @Liripo

This looks like unexpected behavior, possibly a bug. There should be some intergenic reads that are not antisense.
I will look into it shortly.

Cheers
Alex

[alexdobin]

Hi @Liripo

I have fixed this issue, please try this patch:
https://github.com/alexdobin/STAR/releases/tag/2.7.10b_alpha_220111

Now intergenic reads can be counted as
intergenic = genomeU + genomeM - exonic - intronic - exonicAS - intronicAS

[Liripo]

@alexdobin
Thank you so much for your quick help! I'll try it out tomorrow.

[Liripo]

I tried it and indeed the result is different.