rRNA contamination due to GL000220.1

[ramaniak]

We are carrying out RNAseq on some human tissues and have been facing rRNA contamination issues.
We first carried out library prep with the Ribozero and while this depleted most of the cytoplasmic rRNA we noticed that there was still mitochondrial contamination. We therefore switched to the Ribozero-gold and the were happy that the mitochondrial rRNA were also eliminated.

On further QCing on the latest samples we noticed that there were a lot of reads that were now mapping to GL000220.1, an unplaced contig in GRCh37 which has a number of rRNA genes annotated in it. Since this is unplaced we did not check this initially.

My questions are
1. Is anyone else aware of this issue?
2. Are the kits from epicentre expected to remove RNA related to this contig as well?
3. Are there other ways to eliminate them during library prep that does not involve doing polyA?


Any help/suggestions/links would be great. Happy to provide further details if required.

Thanks

[pmiguel]

We are carrying out RNAseq on some human tissues and have been facing rRNA contamination issues.
We first carried out library prep with the Ribozero and while this depleted most of the cytoplasmic rRNA we noticed that there was still mitochondrial contamination. We therefore switched to the Ribozero-gold and the were happy that the mitochondrial rRNA were also eliminated.

On further QCing on the latest samples we noticed that there were a lot of reads that were now mapping to GL000220.1, an unplaced contig in GRCh37 which has a number of rRNA genes annotated in it. Since this is unplaced we did not check this initially.

My questions are
1. Is anyone else aware of this issue?
2. Are the kits from epicentre expected to remove RNA related to this contig as well?
3. Are there other ways to eliminate them during library prep that does not involve doing polyA?


Any help/suggestions/links would be great. Happy to provide further details if required.

Thanks

We did have an issue once where we had a large amount of sequence from the biotynylated probes used to pull out the rRNA. (In this case >60% of the reads were from these probes.) We could tell they were from the kit because our total RNA was actually from cattle, but the rRNA sequence was human/mouse/rat. Also the reads were on the wrong strand (this was a stranded library) for them to be rRNA.

About your "GL000220.1"-mapping reads. GL000220.1 is a 161kb contig and so likely contains quite a bit of non-rRNA gene repetitive DNA. So you may just be looking at Alu's or some other source of transcribed interspersed repeat.

The kit just has a bunch of biotinylated oligos (probes) antisense of various rRNA genes (mitochondrial and nuclear). The magnetic beads are streptavidin conjugated and remove RNA that anneals to the probes. You would expect it to include only genes that Epicentre specifies in their documentation.

--
Phillip