Flexible Method for Targeted Transcript Depletion from RNA-Seq Libraries —
Mouse, Rat, Drosophila, and Arabidopsis
Steve Kain1, Lin Pham1, I-Ching Wang1, Marie Eide1, Dana Burow2, and Doug Amorese1
1
NuGEN Technologies, 201 Industrial Road, San Carlos, CA - USA; 2University of California at Merced, Merced, CA - USA
Fig. 3
1st
strand
synthesis
2nd
strand
synthesis
Adaptor
Ligation
Fragmentation
Gene-specific primers (GSP)
+
Strand
Selection
Polymerase
Enrichment
PCR
InDA-C
+
Cleavage
Site
FWD
InDA-C (Both)
InDA-C
(Chloroplast rRNA)
Cleavage
reagent
InDA-C (rRNA)
FWD
REV
10 ng total RNA
No InDA-C
FWD
100 ng total RNA
FWD
REV
REV
Ribosomal RNA
0
10
20
30
FWD
REV
Fig. 4
t = 0 hr
#1
t = 0 hr
#2 Biological Replicate
100,000
10,000
RPKM t = 0 #2
Assay workflow illustrating targeted depletion of
unwanted transcripts using InDA-C. After adaptor
ligation and strand selection the library is incubated
with gene-specific primers (GSP) which target inserts
containing unwanted transcripts such as rRNA. Primer
extension into the reverse adaptor (REV) creates a
cleavage site in the double-stranded adaptor. Addition
of the cleavage reagent specifically cuts these reverse
adaptors, making them non-amplifiable during
enrichment PCR and cluster formation.
96.94% Other Mapped
3.06% rRNA
100
10
1
0.1
0.01
95.72% Other Mapped
4.28% rRNA
0.001
0.001
~ 7 hours
t = 1 hr
#1
Table 1
t = 1 hr
#2 Biological Replicate
100,000
Organism
Transcripts Targeted for Deletion
Human
Cardiac actin and myosin
RPKM t = 1 #2
10,000
97.48% Other Mapped
2.52% rRNA
rRNA, globin
t = 3 hr
#1
rRNA, housekeeping genes
rRNA, chloroplast RNA
Drosophila
rRNA
Top 30 expressed transcripts in host plant
88.60% Other Mapped
11.40% rRNA
Human host transcripts, TB rRNA
Strand Selection
Mouse
Rat
F O R M O R E I N F O R M AT I O N
To receive information on the design of custom
workflows using InDA-C, or for any other
questions, please contact Steve Kain at
[email protected]
% Aligned
Reads
% rRNA
Reads
% Strand
Retention
(Coding)
Transcripts
Detected
+
90
8.7
99.4
18,439
–
95
80
99.3
15,395
+
94
2.5
98.9
12,098
–
97
53.1
98.8
7,202
+
86
7.6
99.1
14,684
–
96
85.7
98.8
12,003
InDA-C
Targets Unwanted
Transcripts (rRNA)
RNA-Seq library construction workflow. Library
adaptors are specific for the Illumina sequencing
platforms, and enable barcoding up to 96-plex.
100
1000
10,000
100
1000
10,000
100
1000
10,000
Drosophila
r = 0.90
1000
100
10
1
0.1
0.001
0.001
0.01
0.1
1
10
t = 3 hr
#2 Biological Replicate
100,000
92.58% Other Mapped
7.42% rRNA
r = 0.93
1000
100
10
1
0.1
0.01
0.001
0.001
0.01
0.1
1
10
RPKM t = 3 #1
Examples of RNA-Seq projects. Projects used custom
InDA-C primers to target the indicated unwanted
transcripts for depletion from the RNA-Seq library.
PCR
10
0.01
97.29% Other Mapped
2.71% rRNA
RPKM t = 3 #2
Grape
Table 2
Strand-specific
Transcript-depleted
cDNA Library
1
RPKM t = 1 #1
Ligation with Nucleotide
Analog-marked Adaptors
InDA-C Mediated
Adaptor Cleavage
0.1
rRNA, casein protein RNAs
Tuberculosis
End Repair
0.01
RPKM t = 0 #1
Plant fungus
Optional Fragmentation
r = 0.83
1000
10,000
Second Strand Synthesis
with Nucleotide Analog
70
The percentage of informative reads was increased
from ~8.5% to ~57% by targeted depletion using
InDA-C primers; InDA-C primers designed to
cytoplasmic 25S, 18S, 5.8S rRNA + chloroplast rRNA
Primer Extension to Form Cleavage Site
Yeast
Oligo-dT and
Random Priming
60
FWD
Other unwanted
transcripts
Mouse
First Strand Synthesis
50
% Informative Reads
FWD
Goat
10 ng Total RNA
40
REV
Fig. 1
Fig. 2
REV
ABSTRACT
This poster describes a novel method, Insert
Dependent Adapter Cleavage (InDA-C), for
effective removal of specific transcripts from RNASeq libraries without impacting non-targeted
transcripts. InDA-C employs specific and robust
enzymatic steps to eliminate undesirable transcripts
such as rRNA during library construction without
perturbing the original total RNA population as
with hybridization capture methods. The specificity
of transcript depletion relies on InDA-C primers
which can be designed to target virtually any class
of unwanted transcripts from any species. The
library construction workflow uses as little as 10
ng of input total RNA, produces a strand-specific
library, and is highly adaptable for depletion of any
unwanted transcript(s). Here we report the unbiased
removal of rRNA and chloroplast RNA from RNASeq libraries across a variety of important model
organisms including Mouse, Rat, Drosophila and
Arabidopsis. Use of InDA-C primers designed
against cytoplasmic and mitochondrial rRNAs
from these species resulted in the reduction of
sequencing reads derived from these transcripts to
2.5 – 8.7% of the total mapped reads. In the case of
Arabidopsis, the combined use of InDA-C primers
targeting rRNA and chloroplast rRNA resulted in an
increase of informative reads (non-rRNA and nonchloroplast rRNA) from 8.5% in absence of InDA-C
depletion to 57% following targeted depletion
with InDA-C. Therefore, through the use of InDA-C
technology a greater percentage of RNA-Seq
sequencing reads can be directed towards desired
coding and non-coding transcripts.
Sequencing performance metrics. Sequencing
performance metrics with InDA-C primers targeting rRNA
from different Model Organisms. Input is 10 ng total
RNA from Mouse Universal Reference, Rat Brain, and
Drosophila S2 RNAs; The number of detected transcripts
is calculated using an FPKM threshold = 0.1.
www.nugen.com
Measurement of genome-wide mRNA decay in
whole fly embryos by pulse-chase labeling of nascent
transcripts with 4-thiouridine. The results show a
reduction in %rRNA reads to ~3–4% at t=0 hours using
InDA-C, which increases to ~7–11% following the 3-hour
chase to due to mRNA degradation and a relatively
higher %rRNA
CONCLUSIONS
NuGEN has developed a strand-specific RNASeq library construction method that allows for
researchers to customize depletion of unwanted
transcripts:
•Customizable: NuGEN will assist researchers to
design and source custom InDA-C primers for
targeted depletion of unwanted transcripts
•Integrated method for transcript reduction: Uses
targeted depletion during library construction to
minimize undesired sequences in the final library
•A complete solution for strand-specific RNASeq: Includes all required components for
preparation of strand-specific RNA-Seq libraries for
use on all Illumina NGS platforms
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