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Microbial RNA sequencing enabled with theIllumina Ribo-Zero™ Plus rRNA Depletion Kit
Focus on analyzing high-value portions of the transcriptome and save on sequencing costs.

Introduction

Ribosomal RNA depletion

Microbial transcriptomic analysis by RNA sequencing is impeded by
the high relative abundance of ribosomal RNA (rRNA) in bacterial cells,
comprising ≥ 85% of molecules in total RNA.1 Removal of rRNA priorto RNA-Seq enables researchers to focus on analyzing high-value,
informative portions of the transcriptome while lowering sequencing
costs. To assist with rRNA removal, Illumina offers the Ribo-Zero PlusrRNA Depletion Kit.

10–250 ng of total RNA from each sample were processed forenzymatic ribodepletion with the Illumina Ribo-Zero Plus rRNA
Depletion Kit (Illumina, Catalog no. 20037135) (Figure 1). Briefly,
RNA is hybridized with DNA probes complementary to rRNA. Theseprobes target RNase H to digest rRNA species. The DNA probes arethen digested with DNase I, followed by a bead purification of theremaining, intact RNA. For comparison, total RNA from each sample
was also processed with the Ribo-Zero rRNA Removal Kit.2

How it works
rRNA targeting

This new methodology facilitates rich transcriptome analyses ofmicrobial isolates. The single-tube enzymatic ribodepletion method
is compatible with low inputs (≤ 100 ng) and reduces rRNA in bothprokaryotic and eukaryotic samples. Abundant rRNA is removed fromtotal RNA by targeted hybridization to DNA probes and subsequent
RNase H-mediated cleavage. Ribodepleted samples then undergo
library preparation, sequencing, and data analysis.

Hybridization with rRNA-complementary DNA probesrRNA depletion
RNase H digestion of rRNA targeted by DNA probes

DNA probe digestion

This technical note demonstrates rRNA depletion performance on
microbial isolate and community samples with the Illumina RiboZero Plus rRNA Depletion Kit, as compared to the Ribo-Zero kit(previously available with TruSeq™ Stranded Total RNA and TruSeq
Stranded mRNA).

DNase I treatment of DNA probes

Bead cleanup
Removal of enzymes and oligonucleotides

Methods

Library preparation
TruSeq Stranded Total RNA sample prep kit

Sample preparation
Several microbial isolates and mock community standards wereobtained from ATCC for evaluation (Table 1). Total RNA was extractedusing the RNeasy PowerMicrobiome Kit (QIAGEN, Catalog no. 2600050), following the manufacturer’s protocol. Total RNA integrity andquantity were measured using the Agilent Bioanalyzer (Agilent, Catalog
no. G2939BA).
Table 1: Microbial samples tested in this study
Sample type

Microbial species

Microbial isolates

B. cereus, B. subtilis, E. cloacae, E. coli, S. epidermis

ATCC MSA-2002(20-strain even mix)

A. baumannii, A. odontolyticus, B. adolescentis,
B. cereus, B. vulgatus, C. acnes, C. beijerinckii,
D. radiodurans, E. coli, E. faecalis, H. pylori,
L. gasseri, N. meningitidis, P. aeruginosa,
P. gingivalis, R. sphaeroides, S.agalactiae, S. aureus,
S. epidermidis, S. mutans

ATCC MSA-2006(human gut mix)

B. adolescentis, B. fragilis, B. vulgatus, C. difficile,
E. cloacae, E. coli, E. faecalis, F. nucleatum, H. pylori,
L. plantarum, S. enterica, Y. enterococolitica,

Figure 1: Illumina Ribo-Zero Plus ribodepletion workflow.

Library preparation and sequencing
Ribodepleted and nondepleted RNA (control) were prepared forsequencing using the TruSeq Stranded Total RNA LIbrary Prep kit*.3
Libraries were pooled and sequenced on a MiSeq™ or NextSeq™ 550
System, using a 2 × 76 bp paired-end read length.

Data analysis
All sequence filtering, alignment, and transcript coverage wereperformed in BaseSpace™ Sequence Hub using a custom workflow(Figure 2). This analysis workflow is applicable to any microbe with anavailable annotated genome. Transcript counts (> 0.1 transcripts permillion (TPM) generated by the RNA-Seq Alignment App were used
for pairwise linear regression and correlation analysis. To quantifyrRNA from multiple strains within microbial community samples, therespective rRNA sequences (from NCBI annotated genomes) wereused as inputs for the custom analysis workflow.
*For Research Use Only. Not for use in diagnostic procedures.

Indicates recommended library preparation kit; protocol is compatible with other kits.

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Prepare Library | Sequence | Analyze DataResults

Microbial genome sequence and annotation files(FASTA, GTF retrieved from NCBI)

Effective rRNA removal with the Illumina Ribo-Zero Plus
kit across various microbial species

Partition rRNA sequences
Parse rRNA sequences to denote as abundant sequences in analysis

Transcriptome sequencing results of five microbial isolates preparedwith the Illumina Ribo-Zero Plus kit, Ribo-Zero kit, or no ribodepletion
were compared. Ribodepletion with the Illumina Ribo-Zero Plus
kit was highly effective and outperformed the Ribo-Zero kit in allspecies tested, as measured by percent rRNA reads (Figure 3). The
improvement was most significant on a low-input sample (E. coli 10 ng
RNA), with < 0.5% vs 13% average rRNA reads for Illumina Ribo-Zero
Plus and Ribo-Zero, respectively.

RNA Custom Genome Builder App
Create STAR-compatible microbial transcriptome

Microbial sample FASTQs

Significant reduction in minimum sequencing depth fortranscript detection with the Illumina Ribo-Zero Plus kit

RNA-Seq Alignment App
STAR-alignment and salmon-transcript quantification

Figure 2: Microbial RNA-Seq custom analysis workflow—The custom
analysis workflow involves using the RNA Custom Genome Builder App to createa STAR-compatible microbial transcriptome. The RNA-Seq Alignment App isused for STAR-alignment4 and salmon-transcript5 quantification.

To assess the enrichment of biologically important RNA reads with
the Illumina Ribo-Zero Plus kit, the number of transcripts detected at
different read depths was compared with and without ribodepletion(Figure 4). In general, a 20–50× reduction in read depth was needed todetect an equivalent number of transcripts with the Illumina Ribo-ZeroPlus kit. The lower read depth requirement greatly reduces the cost ofsequencing for microbial transcriptome profiling.

Microbial isolate (nondepleted rRNA read %)B. cereus (95.8%)

B. subtilis (94.8%)

E. cloacae (86.7%)

E. coli (94.3%)

S. epidermis (96.1%)

201816% rRNA reads

1413.513.0121086420

3.9
2.9

2.4

2.4
1.4
0.6

Ribo-Zero
Plus
10 ng

Ribo-Zero

100 ngRibo-Zero
Plus
250 ng0.23 0.28

0.03

Ribo-ZeroRibo-Zero
Plus

Ribo-ZeroRibo-Zero
Plus

1.30.2

Ribo-ZeroRibo-Zero
Plus

Ribo-ZerorRNA depletion method

Figure 3: Comparison of ribodepletion methods—Enzymatic ribodepletion was compared against the Ribo-Zero rRNA Removal Kit for rRNA depletion from five
microbial species. For each species, all sample read depths were normalized and the mean percent rRNA reads from two replicates is shown.

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For Research Use Only. Not for use in diagnostic procedures.

Prepare Library | Sequence | Analyze Data

Ribo-Zero Plus

4000

No depletion

B. subtilis

3500

Transcripts detected

3000

2500
Species

4000

3500
E. coli

3000

2500
0.4

0.5

0.6 0.7 0.821345678

920

1030405060

7080 90

100200Sequencing reads (M)Figure 4: Transcript detection at various read depths—The number of transcripts detected with and without enzymatic ribodepletion was compared at various
read depths for B. subtilis and E. coli. Enzymatic ribodepletion resulted in a 20×–50× reduction in read depth needed to detect an equivalent number of transcripts, ascompared to nondepleted sequencing results.

10000030000

E. coli

50000
30000
20000

20000
10000
7000
5000

10000
6000
4000
3000
2000

3000

R2 = 0.999

R2 = 0.993

2000

Replicate 2

Replicate 2B. subtilis

1000
500
300
200
100
60
4030201000700500300200100705010532302011012 3 510 20 401003001000

3000 10000 40000

Replicate 11020 30 50100200 400

1000

300010000

30000

Replicate 1Figure 5: Reproducible transcriptome analysis—A pairwise linear regression of gene expression levels (determined as TPMs) between technical replicates wasperformed. High correlation (R2 > 0.99) was observed for two different microbial isolates, E. coli and B. subtilis.

Reproducible transcriptome sequencing of microbial
species enabled with the Illumina Ribo-Zero Plus kit

Effective rRNA removal in metatranscriptomic samples
with the Illumina Ribo-Zero Plus kit

To demonstrate the reproducibility of RNA-Seq data enabled with
the Illumina Ribo-Zero Plus kit, a pairwise linear regression of gene
expression levels (determined as TPMs) between technical replicates
was performed. High correlation (R2 > 0.99) was observed for two
different microbial isolates, E. coli and B. subtilis (Figure 5).

After demonstrating the effectiveness of the Illumina Ribo-ZeroPlus kit with microbial isolates, ribodepletion was evaluated withmetatranscriptomic samples. Two mock community samples fromATCC (Table 1) were tested with inputs of 10 ng and 80 ng total
RNA. The Illumina Ribo-Zero Plus kit outperformed the Ribo-Zero kit,
reducing the percent rRNA reads from an undepleted level of ~83%
to < 2%, and from ~95% to < 13%, for samples MSA2002 andMSA2006, respectively (Figure 6). The Ribo-Zero kit resulted in lesseffective rRNA removal and also greater variability between replicates(Figure 6).

For Research Use Only. Not for use in diagnostic procedures.

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Prepare Library | Sequence | Analyze Data

Learn more

Mock community
MSA2002

90MSA2006
10 ng
80 ng

80

References

70

1.

60

% rRNA reads

Learn more about the Illumina Ribo-Zero Plus rRNA Depletion Kit, visit
www.illumina.com/products/by-type/accessory-products/ribo-zeroplus-rrna-depletion.html

50

2.
40

3.
36.9

30

32.6

4.
5.

20
12.9

Karpinets TV, Greenwood DJ, Sans CE, Ammons JT. RNA: protein ratio of
the unicellular organism as a characteristic of phosphorous and nitrogenstoichiometry and of the cellular requirement of ribosomes for protein
synthesis. BMC Biol. 2006;4:30.
Illumina (2016).Ribo Zero rRNA Removal Kit Reference Guide(15066012 v02) Accessed March 16, 2020.
Illumina (2013). TruSeq Stranded Total RNA Sample Prep Guide(15031048 E). Accessed March 16, 2020.
Dobin A, Daves CA, Schlesinger F, et al. STAR: ultrafast universal RNA seqaligner. Bioinformatics. 2013;29(1):15–21.
Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides
fast and bias-aware quantification of transcript expression. Nat Methods.
2017;14(4):417–419.

101.110

Ribo-Zero
Plus

Ribo-ZeroRibo-Zero
PlusRibo-ZerorRNA depletion method
Figure 6: Effective ribodepletion in mock communities—The Illumina RiboZero Plus kit outperformed the Ribo-Zero kit with lower, less variable amountsof rRNA present after depletion, as measured by percent rRNA reads for two
different microbial mock communities with different input amounts.

SummaryThe Illumina Ribo-Zero Plus rRNA Depletion Kit is a robust,effective, and simple method to reduce rRNA content for highquality microbial transcriptome analysis. The kit meets or exceeds
Ribo-Zero performance metrics in all the tested microbial isolates
and “microbiome” mock community samples. The Illumina RiboZero Plus kit is uniquely compatible with low inputs, facilitating
metatranscriptomic analysis of low biomass samples.

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com© 2020 Illumina, Inc. All rights reserved. All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark
information, see www.illumina.com/company/legal.html. 1270-2020-003-A QB9679

For Research Use Only. Not for use in diagnostic procedures.

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