Plant total RNA with rRNA depletion: result expectations

[dejonggr]

Hi all,

I recently got some RNA-seq data back that's left a bit puzzled. I opted for total RNA with rRNA depletion during library prep which was then sequenced using the Novaseq platform.

Of the resulting reads, there are some samples where only 30% of reads map to the nuclear genome - should this be expected?

I did some sleuthing and most unmapped reads map to the plastid.

I expected plant chloroplasts to feature prominently (given it is a leaf) but not constitute ~70% of my data.

Has anyone experienced this before? Was there a mistake made along the way?

[nucacidhunter]

They might have used Ribo-Zero for plant seed/root which lacks probes for removing chloroplast transcripts. This kit was discontinued late last year and updated kit is used for all plant tissue types.

[SNPsaurus]

This paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015939/ shows >30% chloroplast-mapped reads in rRNA depleted total RNA prepared from seedlings. Your 70% is high, but a leaf might have more plastid transcription compared to a seedling (just hand waving but it seems possible you are seeing a biologically relevant level of plastid transcription).

[nucacidhunter]

Link to Illumina Ribo-Zero Plant performance: https://www.illumina.com/products/by...l?langsel=/us/

[dejonggr]

I'm still waiting the name of the specific kit that was used for rRNA depletion.

Also, in the meantime I discovered that 30-38% of all reads (sample-wide) mapped to a single gene region (psbA) - which is over 50% of the chloroplastic reads. I tried to do a cursory lit search but I couldn't find any evidence that for such dramatic expression of this gene (but it does make sense given its importance for photosynthesis.