An Improved rRNA Removal Process for
RNA-Seq Library Preparation
Jim Pease, Roy Sooknanan, John Hitchen, and Agnes Radek
Epicentre® (an Illumina® company), 726 Post Road, Madison, WI 53713, USA
Introduction
rRNA Removal from Plant RNA
Figure 2. The Ribo-Zero™ Gold Kit (Human/Mouse/Rat)
removes both cytoplasmic and mitochondrial rRNA.
Current methods for making RNA-Seq libraries typically require removing rRNA
from a total RNA sample prior to library preparation. Existing technologies—such
as oligo(dT)-based poly(A)+ RNA capture methods and selective oligonucleotidebased rRNA removal methods—require the use of high-quality, intact total RNA
samples. Yet, even after two passes, the level of rRNA contamination is often too
high in these final RNA-seq libraries. Additionally, poly(A)+ enrichment results in the
loss of noncoding, non-rRNA sequences that are an important component of the
transcriptome.
We describe a greatly improved hybridization-capture process (Ribo-Zero™
technology) that removes >99% of the cytoplasmic (nuclear-encoded) rRNA and,
optionally, mitochondrial and chloroplast rRNA, from both intact and partially
degraded RNA samples. The reduction in rRNA sequences improves sequence
depth and coverage and increases the percentage of uniquely mapped sequence
reads. The Ribo-Zero process recovers mRNA and large noncoding non-rRNA and
maintains the expression profile of the samples.
Methods
Figure 1. Schematic overview of the Ribo-Zero™ Magnetic process.
Table 3. Removal of plant cytoplasmic and chloroplast rRNA
(qPCR analysis).
0.52%
0.02%
0.24%
0.05%
0.31%
Aligning exomic
6.72%
Partial exomic
24.04%
31.94%
27.40%
28.10%
Genomic
4.48%
12.45%
31.68%
32.05%
Intronic
16S mtrRNA
12S mtrRNA
28S rRNA
Five micrograms of total RNA isolated from MCF-7 cells was treated with
either the standard Ribo-Zero Kit (Human/Mouse/Rat) or the Ribo-Zero Gold
Kit. RNA-Seq libraries were prepared with the using the ScriptSeq™ RNASeq Library Preparation Kit (Epicentre) and sequenced on Illumina® GAIIx
and HiSeq 2000 platforms. The Ribo-Zero Gold Kit reduced the number of
mitochondrial rRNA reads from approximately 7% to <1%. Data courtesy of
Vladimir Benes and Jonathon Blake, EMBL GeneCore, Heidelberg, Germany.
Total RNA
rRNA Removal
(Nuclear)
Input RNA
Sample
25S
18S
rRNA Removal
(Chloroplast)
5.8S
23S
16S
5S
4.5S
Arabidopsis
>99.99% >99.96% >99.94% >99.99% >99.98% >99.97% >99.94%
thaliana leaf
Soybean
root
>99.99% >99.95% >99.99% >99.98% >99.97% >99.75% >99.95%
Soybean
seed
>99.99% >99.99% >99.97% >99.98% >99.98% >99.85% >99.95%
The indicated plant total RNA samples (1-5 µg) were treated using the Ribo-Zero
Plant Kits. The amount of residual rRNA was determined by random-primed
qRT-PCR using PCR primers to the small rRNAs and to multiple regions of the
large rRNAs. Percent rRNA removal was calculated from rRNA-specific ΔCT
values, normalized to those of the EF-1α transcript.
Table 4. Removal of plant cytoplasmic and chloroplast rRNA
(RNA-Seq analysis).
Intact or fragmented (e.g., FFPE)
Add Ribo-Zero™ rRNA
Removal Reagents
Figure 3. High correlation of differential gene expression compared
to MAQC qPCR data.
Incubate 15 minutes
A
Add
Magnetic Beads
rRNA Reads
(Nuclear-Encoded)
rRNA Reads
(Chloroplast)
RNA Sample
25S
18S
5.8S
23S
16S
5S
4.5S
Arabidopsis
thaliana leaf
0.19%
0.49%
0%
0.14%
0.08%
0%
0%
A ScriptSeq™ RNA-Seq library was prepared from 30 ng of Ribo-Zero-treated
RNA and sequenced on an Illumina® GAIIx sequencer. In total, about 1% of
sequencing reads corresponded to rRNA.
Incubate 10 minutes
Remove
Magnetic Beads
Purify rRNA-depleted RNA
(Ethanol precipitation or bead-based)
Table 5. Compatibility of the Ribo-Zero™ Plant Kits.
B
A single-pass Ribo-Zero rRNA removal process takes about 1.5 hours. The
typical yield of rRNA-depleted RNA is 3%-10% of the amount of input total RNA.
Results
rRNA Removal from Eukaryotic RNA
Table 1. RNA-Seq comparison between Ribo-Zero™ technology
and poly(A) enrichment.
RNA
Sample
rRNA
Removal
Method
rRNA Reads
Mapped Reads
Uniquely
Mapped Reads
UHRR
Ribo-Zero
0.04%
87.0%
79.7%
UHRR
Poly(A)+
5.3%
80.7%
72.5%
BrRR
Ribo-Zero
0.04%
88.0%
81.3%
BrRR
Poly(A)+
2.6%
79.7%
74.7%
ScriptSeq™ RNA-Seq libraries were prepared from Universal Human Reference
RNA (UHRR) or human brain (BrRR) RNA after treatment with either the RiboZero rRNA Removal Kit (Human/Mouse/Rat) or poly(A) enrichment. RNA-Seq
libraries made from Ribo-Zero-treated samples produced significantly fewer
rRNA reads and more uniquely mapped reads than poly(A)-enriched RNA.
ScriptSeq™ RNA-Seq libraries were prepared from Ribo-Zero-treated intact (A)
or fragmented (B) UHRR and BrRR total RNA. The high correlation with MAQC
qPCR data indicates that Ribo-Zero treatment does not alter the expression
profiles of the samples.
Table 2. Compatibility of the Ribo-Zero™ Kit (Human/Mouse/Rat)
with other eukaryotic species.
Species
Comments
Human, mouse, rat
>99% removal of 28S, 18S, 5.8S and 5S rRNAs
Other mammalian
(e.g., horse, cow, pig)
Not tested but expected to perform well.
Canine (dog)
Efficient removal of all rRNAs.
Drosophila
Will not remove 5S rRNA or ‘right’ fragment of 28S rRNA.
C. elegans
Use highest quality RNA possible.
Yeast
Removes 26S, 18S, 5.8S rRNAs from intact sample. Will not
remove 5S rRNA. Use highest quality RNA possible.
Xenopus
Use highest quality RNA possible.
Mosquito
Not recommended. Poor removal of 28S and 5S rRNAs.
Zebrafish
Efficient removal of 28S, 18S, and 5S rRNAs. 5.8S rRNA
removal has not been tested.
Lamprey
Use highest quality RNA possible.
Shrimp
Not recommended
Aspergillus niger
300 nt region of 18S rRNA will not be removed from a
degraded sample. Use highest quality RNA possible.
Nematostella vectensis
Use highest quality RNA possible.
Plasmodium falciparum
Removes 28S and 18S rRNA. Will not remove 5.8S or 5S
rRNAs.
Species
Comments
Maize
Efficiently removes cytoplasmic and chloroplast rRNAs
Soy bean
Efficiently removes cytoplasmic and chloroplast rRNAs
Arabidopsis
Efficiently removes cytoplasmic and chloroplast rRNAs
Physcomitrella (moss)
Efficiently removes cytoplasmic and chloroplast rRNAs
Rice
Efficiently removes cytoplasmic and chloroplast rRNAs
Chlamydomonas
Use highest quality RNA possible.
Marsilea vistita
(water clover)
Expected to work well.
Conclusions
 Ribo-Zero kits remove >99% of rRNA from intact and partially degraded total
RNA samples.
 Ribo-Zero-treated RNA samples produce more uniquely mapped RNA-Seq
reads compared to poly(A) enrichment.
 Ribo-Zero treatment recovers mRNA and ncRNA for whole transcriptome
RNA-Seq.
 Ribo-Zero treatment maintains the non-rRNA transcript profile of the original
RNA sample.