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SeqCap RNA Enrichment System
More comprehensive transcriptome, less sequencing
Total RNA
Stranded cDNA Library Preparation
Researchers are currently able to perform whole-transcriptome
sequencing (RNA-Seq) of cDNAs derived from RNA to
evaluate expression levels, variant splicing events, SNPs,
and InDels throughout the transcriptome. But the standard
RNA-Seq approach has significant drawbacks. Because the
whole-transcriptome is largely comprised of highly abundant
transcripts, many rare events are not detected due to the
depth of sequencing necessary to resolve them.
The SeqCap RNA system is designed to overcome these
drawbacks. By helping you target your transcripts of interest
in your transcriptome of interest, this system offers
unprecedented discovery power.
Hybridization
Benefits of the SeqCap RNA system:
Probes
n Discover with more confidence: Achieve unprecedented
depth and sensitivity for rare transcript detection with
hundreds to thousands fold of high-fidelity enrichment.
Capture and Washing
Washing
Amplification and QC
n Broaden your research scope: Maximize the discovery
and characterization of novel exons, splicing variants,
mutations and gene fusions through the ability to
target your transcripts of interest with up to 200 Mb
custom designs.
n Experience improved efficiency: Dramatically increase
your sample capacity with the same amount of sequencing
through optimized workflow.
The SeqCap RNA system comes with the most comprehensive
lncRNA (long non-coding RNA) design as the fixed content and
a broad range of custom offerings to meet your research needs.
Learn more about these advantages at
www.nimblegen.com/SeqCapRNA
Sequencing
For life science research only.
Not for use in diagnostic procedures.
Effective answer to the RNA-Seq challenge
The challenge with standard RNA-Seq is the
overwhelming number of sequencing reads that derive
from a small subset of genes. Relatively few of the reads
are from the bottom 50% of genes as measured by
transcript abundance (see Figure 1). The end result is
limited sensitivity to low-abundance transcripts while
wasting most sequencing resources.
This challenge can be overcome by targeting the
transcripts with NimbleGen’s proprietary sequence
capture technology. The SeqCap RNA system can focus
sequencing resources on a subset of genes, enrich rare
transcripts, and enable the search for putative transcripts
in target genomic regions. The improved utilization of
sequencing allows for the processing of more samples
and obtaining data more quickly, while reducing cost.
Figure 2 shows a comparison of standard RNA-Seq to
SeqCap RNA reads from Human Brain Reference RNA.
As shown, 2.8 million SeqCap RNA reads provide the
same detection sensitivity as 142.8 million standard RNASeq reads. This means that this SeqCap RNA design is
significantly more efficient, requiring 50 times fewer reads
to achieve sensitivity to similar abundance transcripts. The
capture enrichment in the experiment also shows that this
improvement in targeted sequencing does not negatively
impact the ability to report transcript abundance.
A
B
p
Figure 1: The challenge of abundant transcripts. RNA-Seq
data generated from ribo-depleted Human Brain Reference RNA
finds that the 1% most abundantly expressed genes account for
approximately 69% of sequencing reads. Data from 142.8 M
paired-end reads were used to generate this figure.
p Figure 2: Performance in preserving transcript abundance
and sensitivity to rare transcripts as measured with ERCC
(External RNA Controls Consortium) spike-in controls.
RNA-Seq (A) vs. SeqCap RNA (B) demonstrates that capture
preserves transcript abundance. Captures were performed with
a 1.8 Mb design targeting 256 genes associated with neurological
disorders and 83 ERCC control transcripts. Standard RNA-Seq
experiment used ribo-depleted input RNA while the SeqCap RNA
experiment used total RNA.
2|
Make a quantum leap in your discovery power
It’s easy to understand that the ability to discover
dramatically more variants is the prominent benefit the
SeqCap RNA workflow has over standard RNA-Seq.
Results for isoform detection using the lncRNA design are
shown in Figures 3 and 4. The results are presented two
ways. In Figure 3, the SeqCap RNA workflow detected 5
to 20 times more lncRNA isoforms, exceeding the FPKM
cutoff values from RNA-Seq, and indicating enhanced
sensitivity to a greater number of transcripts isoforms.
Figure 4 shows that in both experiments, SeqCap RNA
data subsampled to 1 M reads detected more transcript
isoforms than RNA-Seq data subsampled to 20 M reads,
indicating >20 times increased discovery power or
sequencing throughput.
One important application of target enrichment in RNA-Seq
is tissue-specific enrichment designs. Such designs allow
researchers to overcome the negative impact of tissue-specific
top expressors and identify significantly more low-abundance
but differentially expressed transcripts. Figure 5 shows how
this can be done systematically with the SeqCap RNA system,
starting with a standard RNA-Seq step.
A
p
B
Figure 4: Greater isoform discovery with SeqCap RNA. Ambion
Human Kidney Total RNA (A) and Human Liver Total RNA (B) were
sequenced with standard RNA-Seq (black) and SeqCap RNA (red) to
determine the number of lncRNA transcript isoforms detected when
subsampled to various number of reads (x-axis). Captures were
performed with the SeqCap lncRNA Enrichment Kit.
q Figure 5. Overview of tissue-specific enrichment design.
1 RNA sample
A Poly A enrich or ribo-deplete total RNA
BSynthesize cDNA library
2 RNA-Seq
A Sequence cDNA library
3 Primary data analysis
A
B
A Trim and align RNA-Seq reads
B Quantify gene expression
4 Identify capture targets
ASort genes by gene expression (e.g. FPKM)
B Identify genes responsible for top 80% of
reads (adjust percent read cutoff for your
sample type, if appropriate)
C
Identify other genes for exclusion (e.g.
rRNA, mitochondrial, ribosomal proteins)
p
Figure 3: Greater isoform sensitivity with SeqCap RNA. Ambion
Human Kidney Total RNA (A) and Human Liver Total RNA (B) were
sequenced with standard RNA-Seq (blue) and SeqCap RNA (red) to
determine the number of lncRNA (long noncoding RNA) transcript
isoforms with FPKM values exceeding 4 different cutoffs. Captures
were performed with the SeqCap lncRNA Enrichment Kit. All
sequencing subsampled to 20 M reads.
5 Create capture design
A
Provide gene identifiers or coordinates for
gene list that exclude highly expressed and
other genes previously identified
6 Capture and sequence
|3
Achieve workflow simplicity and efficiency
The SeqCap RNA workflow is optimized
to provide multiple advantages:
n Working over a wide range of RNA input amounts, including as low as
10 ng of total RNA, while maintaining linearity of transcript abundance
n Retaining accurate strand origin information with the Kapa stranded
cDNA library construction solution
n Eliminating the need for costly and time-consuming rRNA depletion
step standard in RNA-Seq experiments
n Supporting pre-capture multiplexing for increased efficiency
and reduced cost per sample
n Allowing for detection of challenging GC-rich and low
abundance transcripts
See what others can’t see in RNA-Seq data, and see it clearly,
with the SeqCap RNA Enrichment System.
Ordering Information
SeqCap lncRNA Design:
Comprehensive design for
maximal coverage
lncRNA annotation from two
data sources:
nHuman Body Map catalog
lincRNA (long-intergenic
noncoding RNAs) track in
UCSC genome browser.
Cabili et al. (2011)
Genes Dev. 25: 1915.
RNA-seq dataset of ~4 billion
reads across 24 tissues and
cell types.
Includes lincRNAs and TUCPs
(transcripts with uncertain
coding potential).
nGENCODE v19
Product
Cat. No.
SeqCap lncRNA Enrichment Kit
07 277 270 001
4 reactions
07 277 288 001
48 reactions
07 277 296 001
384 reactions
07 277 300 001
12 reactions
(hg19) representing 32,808 lncRNA
07 279 078 001
48 reactions
and TUCP transcript isoforms.
07 279 086 001
348 reactions
07 279 183 001
12 reactions
07 279 191 001
48 reactions
07 279 205 001
348 reactions
07 279 213 001
12 reactions
07 279 221 001
48 reactions
07 279 230 001
348 reactions
07 277 261 001
24 reactions
07 277 253 001
96 reactions
SeqCap RNA Choice Enrichment Kit
Target up to 7 Mb of genomic regions
SeqCap RNA Choice XL Enrichment Kit
Target 7 – 100 Mb of genomic regions
SeqCap RNA Developer Enrichment Kit
Target up to 200 Mb of genomic regions
Individual Kits and Reagents
KAPA Stranded RNA-Seq Library Preparation
Pack Size
Data presented in this document was processed by Roche NimbleGen unless
otherwise stated. For patent license limitations for individual products please refer to:
www.technical-support.roche.com.
For life science research only. Not for use in diagnostic procedures.
NIMBLEGEN and SEQCAP are trademarks of Roche.
All other product names and trademarks are the property of their respective owners.
Design captures 17.35 Mb region
Published by:
Roche NimbleGen, Inc
500 S. Rosa Rd
Madison, WI
USA
www.nimblegen.com
© 2014 Roche NimbleGen, Inc.
All rights reserved.
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