KAPA RNA HYPER PREP KITSKAPA Hyper Prep with RiboErase (HMR) CV : 0.65 TruSeq S tranded with...
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For Research Use Only. Not for use in diagnostic procedures. Data on file. KAPA RNA HYPER PREP KITS The KAPA RNA HyperPrep Kits utilize novel chemistry that enables the combination of enzymatic steps and fewer reaction purifications, resulting in a truly streamlined solution for the preparation of high-quality RNA-seq libraries. The strand-specific workflow is flexible—supporting library construction from lower-input amounts and degraded samples—and is compatible with both mRNA capture and ribosomal depletion. Kits contain all reagents required for RNA enrichment (if performed) and library preparation, with the exception of KAPA Adapters (available separately). Single-Day RNA. Benefits include: • single-day library construction, inclusive of RNA enrichment • higher success rates with low-input and degraded samples • robust performance across different sample types and input amounts • KAPA Pure Beads for reaction purifications
Transcript of KAPA RNA HYPER PREP KITSKAPA Hyper Prep with RiboErase (HMR) CV : 0.65 TruSeq S tranded with...
For Research Use Only. Not for use in diagnostic procedures. Data on file.
KAPA RNA HYPER PREP KITS
The KAPA RNA HyperPrep Kits utilize novel chemistry that enables the combination of enzymatic steps and fewer reaction purifications, resulting in a truly streamlined solution for the preparation of high-quality RNA-seq libraries. The strand-specific workflow is flexible—supporting library construction from lower-input amounts and degraded samples—and is compatible with both mRNA capture and ribosomal depletion. Kits contain all reagents required for RNA enrichment (if performed) and library preparation, with the exception of KAPA Adapters (available separately).
Single-Day RNA.
Benefits include:
• single-day library construction, inclusive of RNA enrichment
• higher success rates with low-input and degraded samples
• robust performance across different sample types and input amounts
• KAPA Pure Beads for reaction purifications
For Research Use Only. Not for use in diagnostic procedures. Data on file. 2
Single-tube, Single-day Library Prep• Reduce hands-on and overall time through fewer enzymatic and reaction cleanup steps
• Produce strand-specific, sequencing-ready libraries from input RNA in approximately 4 hours
• Complete entire workflow—inclusive of mRNA capture or ribosomal depletion—in a standard workday
• Achieve high throughput and consistency with an automation-friendly workflow
TruSeq Stranded RNA Library Prep Workflow
NEBNext Ultra Directional RNA
Library Prep WorkflowKAPA RNA
HyperPrep Workflow
Fragmentation and Priming
1st Strand cDNA Synthesis
KAPA Pure Beads Cleanups
Adapter Ligation(adapters sold separately)
KAPA Pure Beads Cleanup
2nd Strand Synthesis and A-tailing
Library Amplificationwith KAPA HiFi
Sing
le T
ube
Fragmentation and Priming
1st Strand cDNA Synthesis(not all reagents supplied)
A-tailing
Bead Cleanup(reagents not supplied)
Library Amplification
Bead Cleanups(reagents not supplied)
Bead Cleanup(reagents not supplied)
~6.0 hr ~5.5 hr~4.0 hr
2nd Strand Synthesis
Adapter Ligation
Tube
1Tu
bes
2 an
d 3
Fragmentation and Priming
1st Strand cDNA Synthesis(not all reagents supplied)
A-tailing
Bead Cleanup(reagents not supplied)
Library Amplification
Bead Cleanups(reagents not supplied)
Bead Cleanup(reagents not supplied)
2nd Strand Synthesis
Adapter Ligation(adapters sold separately)
Tube
1Tu
bes
2 an
d 3
Streamlined RNA library preparation. The KAPA RNA HyperPrep workflow reduces overall library preparation time by 1.5 to 2 hours, in comparison to competitor workflows, making library construction possible in a single workday. Additionally, the reduction in the total number of enzymatic and reaction cleanup steps reduces the hands-on time required.
KAPA RNA HyperPrep Workflow
TruSeq Stranded RNA Library Prep Workflow
NEBNext Ultra Directional RNA Library Prep Workflow
For Research Use Only. Not for use in diagnostic procedures. Data on file. 3
Flexible Workflow Options…• Use the KAPA RNA HyperPrep Kit as a standalone workflow, or combine with either the mRNA capture or
KAPA RiboErase (HMR) ribosomal RNA depletion modules
KAPA RNA HyperPrep Kits with RiboErase (HMR) KAPA mRNA HyperPrep KitsKAPA RNA HyperPrep Kits
with RiboErase
Total RNA(25 ng – 1 µg)
Hybridize RNA
DNase Digestion
KAPA Pure Beads Cleanup
KAPA Pure Beads Cleanup
KAPA RNA HyperPrep Workflow
Deplete rRNA with RNase H
Total library prep time:
~2.5 hr
~4.0 hr
~6.5 hr
KAPA mRNAHyperPrep Kits
2nd mRNA Capture
Total RNA(50 ng – 1 µg)
1st mRNA Capture
KAPA RNA HyperPrep Workflow
Total library prep time:
~1.5 hr
~4.0 hr
~5.5 hr
KAPA RiboErase (HMR). Sequencing of rRNA-depleted total RNA samples provides a more comprehensive representation of the whole transcriptome. rRNA is targeted and depleted enzymatically using DNA probes and RNase H resulting in improved coverage of transcripts of interest, including precursor mRNAs and important regulatory species, such as noncoding RNAs.
mRNA Capture. Sequencing of mRNA-enriched samples provides a focused view of the protein-coding regions in the transcriptome. mRNA capture beads are used prior to library preparation with the KAPA RNA HyperPrep workflow, which enriches for mRNA over non-polyadenylated species, such as ribosomal, precursor, and noncoding RNAs.
Enable a Variety of Strand-specific Applications • Input less starting material than other commercially-available workflows
• Generate high-quality libraries—even with degraded samples, such as FFPE
KAPA RNA HyperPrep Kits
KAPA RNA HyperPrep Kits with RiboErase (HMR)
KAPA mRNA HyperPrep Kits
RNA Enrichment None rRNA Depletion Poly(A) Selection
Input Amount 1 – 100 ng into library prep 25 ng – 1 µg into rRNA depletion 50 ng – 1 µg into mRNA capture
Sample TypeHigh-quality total RNA
Degraded or FFPE total RNAPreviously enriched RNA
High-quality total RNADegraded or FFPE total RNA High-quality total RNA
Species Eukaryotic (animal, plant, etc.)Prokaryotic (bacterial, etc.) Human, mouse, and rat Eukaryotic (animal, plant, etc.)
Differentiating Applications Whole transcriptome Non-coding RNA
Whole transcriptome mRNA-Seq
Shared Applications
Gene expression analysis; detection of gene fusions, isoforms, and other structural variants; novel transcript identification; SNV discovery
A workflow to meet a variety of needs. The KAPA RNA HyperPrep workflow is available in three formats: with mRNA capture, with KAPA RiboErase (HMR) for rRNA depletion, or with no RNA enrichment reagents. This flexibility allows users to select the workflow that best meets the needs of their specific application.
For Research Use Only. Not for use in diagnostic procedures. Data on file. 4
22,000
21,700
134,000
132,000
130,000
Gene
s D
etec
ted
Tran
scrip
ts D
etec
ted
22,300
KAPA mRNAHyperPrep
TruSeq Stranded mRNA
NEBNext UltraDirec�onal withmRNA Capture
21,000
18,000
135,000
125,000
115,000
Gene
s D
etec
ted
Tran
scrip
ts D
etec
ted
24,000
KAPA RNA HyperPrep with RiboErase
TruSeq StrandedTotal RNA with Ribo-Zero Gold
NEBNext Ultra Direc�onal withrRNA Deple�on
Sequence What Matters• Waste fewer reads due to the combination of rRNA carryover and PCR duplicates
• Identify more unique transcripts and genes with equivalent sequencing
40
20
10
0
Perc
enta
ge
30
rRNA Duplicates
KAPA RNA HyperPrep with RiboErase
TruSeq StrandedTotal RNA with Ribo-Zero Gold
NEBNext Ultra Direc�onal withrRNA Deple�on
30
20
10
0
Perc
enta
ge
rRNA Duplicates
KAPA mRNA HyperPrep TruSeq StrandedmRNA
NEBNext UltraDirec�onal withmRNA Capture
Better utilize sequencing capacity. The KAPA RNA HyperPrep workflows result in a reduction in the total number of reads wasted due to both PCR duplicates and alignments to rRNA loci (A and B). With an equivalent amount of sequencing, more genes and unique transcripts are identified using the KAPA workflows in comparison to the TruSeq® and NEBNext kits (C and D). Libraries were generated in quadruplicate with 25 ng (rRNA depletion) and 50 ng (mRNA capture) of high-quality Universal Human Reference (UHR) RNA using the manufacturers’ standard recommendations per workflow, where possible. For this and all subsequent data, sequencing was performed using an Illumina® HiSeq® 2500 in high output mode with v4 chemistry and 2 x 100 bp read length. Reads aligning to rRNA were removed and paired reads were randomly subsampled to 14M for comparative analyses, including marked duplicates.
rRNA Depletion mRNA CaptureA
C D
B
For Research Use Only. Not for use in diagnostic procedures. Data on file. 5
KAPA mRNA HyperPrep
NEBNext Ultra Directional with mRNA Capture
TruSeq Stranded mRNA
63,740,000 bp 63,741,000 bp 63,742,000 bp 63.743,000 bp
Coverage
Junctions
Coverage
Junctions
Coverage
Junctions
ENST00000467211 ENST00000266077 ENST00000493772 ENST00000473157 SLC2A4RG
3,641 bp
20
KAPA RNA Hyper Prep mRNA
Illumina 50ng
NEB 50ng
Homo Sapiens
63,741,000 bp63,740,000 bp 63,742,000 bp 63,743,000 bp
3,639 bb
ENST00000467211 ENST00000266077 SLC2A4RGENST000004937772 ENST00000473157
GC ContentGene
YBX1
[0 - 470]
[0 - 470]
42,684 kb 42,686 kb 42,688 kb 42,690 kb 42,692 kb 42,694 kb 42,696 kb 42,698 kb 42,700 kb 42,702 kb42,682 kb
19 kb
ENST00000616651
KAPA RNA HyperPrep with RiboErase (HMR)
NEBNext Ultra Directional with rRNA Depletion
TruSeq Stranded Total RNA with Ribo-Zero Gold
GC ContentGene
[0 - 470]
[0 - 120]
[0 - 120]
[0 - 120]
A
B
Achieve Superior Coverage Uniformity• Obtain more uniform distribution of reads across transcripts
• Improve coverage of difficult GC-rich regions
Improved coverage uniformity. Increased coverage of GC-rich regions (outlined in red) of the YBX1 (A) and SLC2A4RG (B) genes is demonstrated using the Kapa workflows. For the top 1000 transcripts, the KAPA RNA HyperPrep workflows result in more even coverage across transcript lengths, as assessed by both a normalized coverage plot and coverage coefficient of variation (CV), in comparison to competitor workflows (C and D). Libraries were generated with 25 ng (rRNA depletion) and 50 ng (mRNA capture) of high-quality UHR RNA using the manufacturers’ standard recommendations per workflow, where possible.
rRNA Depletion
mRNA Capture
C D
Normalized position along transcript 5' – 3'
1.2
1.0
0.4
0.0
Norm
alize
d Co
vera
ge
1.6
1.4
0.8
0.2
0.6
20 40 60 80 1000
KAPA HyperPrep with RiboErase (HMR) CV: 0.65TruSeq Stranded with Ribo-Zero Gold CV: 0.69NEBNext Ultra Directional with rRNA Depletion CV: 0.69
KAPA mRNA HyperPrep CV: 0.64TruSeq Stranded mRNA CV: 0.68NEBNext Ultra Directional with mRNA Capture CV: 0.69
Normalized position along transcript 5' – 3'
1.2
1.0
0.4
0.0
Norm
alize
d Co
vera
ge
1.6
1.4
0.8
0.2
0.6
20 40 60 80 1000
Normalized position along transcript 5' – 3'
1.2
1.0
0.4
0.0
Norm
alize
d Co
vera
ge
1.6
1.4
0.8
0.2
0.6
20 40 60 80 1000
KAPA HyperPrep with RiboErase (HMR) CV: 0.65TruSeq Stranded with Ribo-Zero Gold CV: 0.69NEBNext Ultra Directional with rRNA Depletion CV: 0.69
KAPA mRNA HyperPrep CV: 0.64TruSeq Stranded mRNA CV: 0.68NEBNext Ultra Directional with mRNA Capture CV: 0.69
Normalized position along transcript 5' – 3'
1.2
1.0
0.4
0.0
Norm
alize
d Co
vera
ge
1.6
1.4
0.8
0.2
0.6
20 40 60 80 1000
rRNA Depletion mRNA Capture
For Research Use Only. Not for use in diagnostic procedures. Data on file. 6
0
100
75
50
25
0
Perc
enta
ge
rRNADuplicates
rRNADuplicates
Perc
enta
ge
100
75
50
25
NEBNext UltraDirectional withrRNA Depletion
TruSeq StrandedTotal RNA with Ribo-Zero Gold
KAPA RNA HyperPrep with RiboErase
KAPA RNA HyperPrep with RiboErase
TruSeq StrandedTotal RNA withRibo-Zero Gold
NEBNext UltraDirectional withrRNA Depletion
Generate High-quality Libraries from Degraded Samples• Input as little as 25 ng with FFPE samples, depending on total RNA quality
• Achieve low duplication rates and highly efficient, reproducible rRNA removal with degraded samples
• Identify more unique transcripts and genes with equivalent sequencing
4,000 [nt]2,0001,00050020025
40
30
20
10
0
Fluo
resc
ence
Thyroid FFPERIN: 2.2Dv200: 47%
[FU]
Fluo
resc
ence
4,000 [nt]2,0001,00050020025
40
30
20
10
0
Duodenum FFPERIN: 2.5Dv200: 29%
[FU]
0
100
75
50
25
0
Perc
enta
ge
rRNADuplicates
rRNADuplicates
Perc
enta
ge
100
75
50
25
NEBNext UltraDirectional withrRNA Depletion
TruSeq StrandedTotal RNA with Ribo-Zero Gold
KAPA RNA HyperPrep with RiboErase
KAPA RNA HyperPrep with RiboErase
TruSeq StrandedTotal RNA withRibo-Zero Gold
NEBNext UltraDirectional withrRNA Depletion
Total RNA electropherograms for two FFPE samples. The thyroid FFPE sample (RIN: 2.2) is higher-quality, with 47% of the RNA measuring >200 nt. In contrast, the duodenum FFPE sample (RIN: 2.5) is lower-quality, with 29% of the RNA measuring >200 nt. Electropherograms were generated using an Agilent® RNA 6000 Pico Kit.
Better utilize sequencing capacity. Using the two FFPE RNA samples shown above, the KAPA RNA HyperPrep Kit with RiboErase (HMR) results in a reduction in the total number of reads wasted due to both PCR duplicates and alignment to rRNA loci in comparison to competitor workflows (A and B). With equivalent sequencing, more genes and transcripts are identified using the KAPA workflow in comparison to competitors (C and D). Libraries were generated in duplicate using 25 ng for thyroid libraries and a minimum of 100 ng for duodenum libraries, due to the lower quality of the duodenum starting material.
A B
C D
4,000 [nt]2,0001,00050020025
40
30
20
10
0
Fluo
resc
ence
Thyroid FFPERIN: 2.2Dv200: 47%
[FU]
Fluo
resc
ence
4,000 [nt]2,0001,00050020025
40
30
20
10
0
Duodenum FFPERIN: 2.5Dv200: 29%
[FU]
KAPA RNA HyperPrep
with RiboErase
TruSeq Stranded Total RNA with Ribo-Zero Gold
NEBNext Ultra Directional with rRNA Depletion
125,000
105,000
85,000
Gene
s D
etec
ted
Tran
scrip
ts D
etec
ted
19,700
17,200
22,000
KAPA RNA HyperPrep
with RiboErase
TruSeq Stranded Total RNA with Ribo-Zero Gold
NEBNext Ultra Directional with rRNA Depletion
19,700
17,200
125,000
105,000
85,000
Gene
s D
etec
ted
Tran
scrip
ts D
etec
ted
22,000
KAPA RNA HyperPrep
with RiboErase
TruSeq Stranded Total RNA with Ribo-Zero Gold
NEBNext Ultra Directional with rRNA Depletion
125,000
105,000
85,000
Gene
s D
etec
ted
Tran
scrip
ts D
etec
ted
19,700
17,200
22,000
KAPA RNA HyperPrep
with RiboErase
TruSeq Stranded Total RNA with Ribo-Zero Gold
NEBNext Ultra Directional with rRNA Depletion
19,700
17,200
125,000
105,000
85,000
Gene
s D
etec
ted
Tran
scrip
ts D
etec
ted
22,000
Higher-quality FFPE Lower-quality FFPE
For Research Use Only. Not for use in diagnostic procedures. Data on file. 7
Achieve Reliable Results with Degraded Inputs• Attain a high degree of expression correlation between paired FFPE and fresh frozen samples,
providing increased confidence in sequence data accuracy
High level of agreement between paired FFPE and fresh frozen expression data, in transcripts per million (TPM). Pearson correlation coefficients show a higher degree of agreement with the KAPA RNA HyperPrep Kit with RiboErase (HMR) in comparison to the TruSeq® Stranded Total RNA Library Prep with Ribo-Zero Gold and NEBNext Ultra Directional Library Preparation with the rRNA Depletion Kits. Libraries were generated in duplicate using 100 ng inputs of paired FFPE-derived and fresh frozen breast tumor total RNA samples using the manufacturers’ standard recommendations per workflow.
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.896
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.820
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.923
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.896
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.820
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.923
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.896
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.820
Fresh Frozen (TPM+1)
FFFE
(TPM
+ 1
)
11
10
100
1,000
10,000
10 100 1,000 10,000
R2 = 0.923
KAPA RNA HyperPrep Kit with RiboErase (HMR)
TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold
NEBNext Ultra Directional Library Prep Kit with rRNA Depletion
KapaBiosystemsKapaBiosystems@KapaBiosystems
© 2016 Roche Sequencing Solutions, Inc. KAPA is a trademark of Roche. All trademarks are the property of their respective owners. A012 10/16For Research Use Only. Not for use in diagnostic procedures. Data on file.
Published by:
Roche Sequencing Solutions4300 Hacienda DrivePleasanton, CA 94588www.kapabiosystems.com
Ordering Information for KAPA RNA HyperPrep KitsRoche Cat. No. Kapa Code Description Kit Size
08098093702 KK8540 KAPA RNA HyperPrep Kit 24 reactions
08098107702 KK8541 KAPA RNA HyperPrep Kit 96 reactions
08098115702 KK8580 KAPA mRNA HyperPrep Kit 24 reactions
08098123702 KK8581 KAPA mRNA HyperPrep Kit 96 reactions
08098131702 KK8560 KAPA RNA HyperPrep Kit with RiboErase (HMR) 24 reactions
08098140702 KK8561 KAPA RNA Hyper Prep Kit with RiboErase (HMR) 96 reactions
Ordering Information for KAPA AdaptersRoche Cat. No. Kapa Code Description Kit Size
08005699001 KK8700 KAPA Single-Indexed Adapter Kit, Set A + B (30 µM) 24 adapters x 40 µL each
08005702001 KK8701 KAPA Single-Indexed Adapter Kit, Set A (30 µM) 12 adapters x 40 µL each
08005729001 KK8702 KAPA Single-Indexed Adapter Kit, Set B (30 µM) 12 adapters x 40 µL each
08005770001 KK8710 KAPA Single-Indexed Adapter Kit, Set A + B (1.5 µM) 24 adapters x 40 µL each
08005788001 KK8711 KAPA Single-Indexed Adapter Kit, Set A (1.5 µM) 12 adapters x 40 µL each
08005796001 KK8712 KAPA Single-Indexed Adapter Kit, Set B (1.5 µM) 12 adapters x 40 µL each
