Data on file.For Research Use Only. Not for use in diagnostic procedures.

MORE COMPREHENSIVETRANSCRIPTOME COVERAGE

KAPA Stranded RNA-Seq Kits with RiboErase (HRM) offer a comprehensive solution for high-quality transcriptome sequencing. A targeted enzymatic strategy offers superior depletion of ribosomal RNA (rRNA), and a more complete representation of the transcriptome, including precursor mRNAs and non-coding RNA. Kits contain KAPA HiFi for high-efficiency, low-bias library amplification, and employ a streamlined, “with-bead” protocol to enable optimal library construction efficiency.

Benefits• Up to 99.98% rRNA depletion of human, mouse or rat rRNA from various sample types

• Compatible with degraded inputs, including FFPE

• Improved detection of noncoding and precursor transcripts

• Robust and reproducible performance across a wide range of input amounts (100 ng - 1 μg total RNA)

• Improved coverage of GC-rich and low abundance transcripts

• Qualified automation methods

• Complete RNA-Seq library prep solution with KAPA Adapters and KAPA Pure Beads

KAPA Stranded RNA-Seq Kits with RiboEraseEvolved to focus

2Data on file.For Research Use Only. Not for use in diagnostic procedures.

Industry-leading rRNA depletion• Superior rRNA depletion from both high-quality and FFPE samples

• More efficient rRNA depletion translates to improved sequencing economy, or deeper coverage of transcripts of interest from the same amount of sequencing

Higher coverage uniformity• More uniform distribution of reads across transcripts

• Minimal 5' - 3' bias

Highly efficient rRNA depletion from both high-quality and challenging samples. In the graph on the left, duplicate libraries were prepared from various inputs of Universal Human Reference (UHR) RNA, with the KAPA Stranded RNA-Seq Kit with RiboErase, or the Illumina® TruSeq® Stranded Total RNA workflow with Ribo-Zero® Gold Kit. In the graph on the right, duplicate libraries were prepared from 100 ng of UHR RNA or kidney FFPE RNA (RIN <3) using the KAPA Stranded RNA-Seq Kit with RiboErase, or the NEBNext® rRNA Depletion Kit. Residual RNA levels are expressed as a percentage of total mapped reads.

Improved coverage of the whole transcript. Coverage and splice junction tracks for the housekeeping beta-actin gene. Improved coverage of both 5'- and 3'- regions was observed in libraries prepared from 100 ng UHR RNA, using the KAPA Stranded RNA-Seq Kit with RiboErase (green). Poor 3'-end coverage was observed for libraries prepared from the same sample and input with the Illumina TruSeq Stranded Total RNA workflow with Ribo-Zero Gold Kit (orange), whereas coverage for the 5'-region was sub-optimal for libraries prepared with the NEBNext rRNA Depletion Kit (blue). Coverage for libraries prepared from undepleted total RNA samples is included as a reference (red).

Minimal positional bias. Minimal 5' - 3' coverage bias was observed in libraries prepared from an undepleted 100 ng UHR RNA sample (red), or with the KAPA RiboErase (green) or Illumina TruSeq Ribo-Zero (orange) workflows. The NEBNext rRNA Depletion workflow resulted in poorer coverage of the 3'-regions of transcripts.

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

KAPA RiboErase vs. Illumina Ribo-Zero Gold KAPA RiboErase vs. NEBNext rRNA Depletion Kit

UHR UHR vs. FFPE Kidney (100 ng)

Res

idua

l rR

NA

(%

)

Res

idua

l rR

NA

(%

)

0.5

0.4

0.3

0.2

0.1

025 ng UHR UaHR FFPE Kidney FFPE Kidney25 ng 100 ng 100 ng 1000 ng 1000 ng

KAPA KAPAIllumina NEB

ACTB

7

p22.2 p21.3 p21.2 p15.3 p15.1 p14.2 p13 p12.2 p11.2 q11.1 q11.22 q21.11 q21.12 q21.3 q22.1 q22.3 q31.1 q31.2 q31.32 q32.1 q33 q34 q35 q36.1 q36.3

3,649 bp

[0 - 5473]5,567,000 bp 5,568,000 bp 5,569,000 bp 5,570,000 bp

[0 - 4870]

[0 - 4279]

[0 - 3376]

KA

PA10

0 ng

Illum

ina

100

ngN

EB10

0 ng

Tota

l R

NA

Coverage

Junctions

Coverage

Junctions

Coverage

Junctions

Coverage

Junctions

GC ContentGene

0 20 40 60 80 1000.0

0.5

1.0

1.5

5' - 3' Transcript length (%)

Nor

mal

ized

mea

n co

vera

ge

Illumina Ribo-Zero GoldNEBNext rRNA DepletionKapa RiboEraseTotal RNA

3Data on file.For Research Use Only. Not for use in diagnostic procedures.

High-quality sequence data• Detect more genes and unique transcripts

• Clearer identification of splice junctions

• Improved coverage of difficult (e.g. GC-rich and low-abundance) transcripts

Improved coverage of GC-rich transcripts. Coverage and splice junction tracks for the hemoglobin-alpha transcript (HBA1), which has an overall GC-content of 62.8%. The GC-content track at the bottom of the image depicts GC-rich regions in red, and AT-regions in blue. Coverage uniformity and splice junction recognition for libraries prepared from 100 ng UHR RNA with the KAPA RiboErase (green) and NEBNext workflows (blue) compared very well with data for libraries prepared from undepleted RNA (red). In contrast, the Illumina TruSeq Ribo-Zero® Gold workflow yielded significantly lower coverage of some of the GC-rich regions, leading to inaccurate assignment of the splice junction.

Better characterization of low-abundance transcripts. Coverage and splice junction tracks for MALAT-1, a low-abundance, long, non-coding RNA transcript. The region shown here is more comprehensively covered in libraries prepared from 100 ng UHR RNA with the KAPA RiboErase workflow (green), than with the Illumina TruSeq Ribo-Zero Gold (orange) and NEBNext rRNA Depletion (blue) workflows. Coverage and splice junction assignment for libraries prepared from undepleted RNA (red) are included for comparison.

Higher quality sequence data. Duplicate libraries were prepared from either 1 μg or 100 ng UHR RNA (as indicated), using each kit according to the manufacturer's standard recommendations. With similar mapping rates, the KAPA RiboErase workflow yielded lower residual rRNA levels and duplication rates, and higher coverage uniformity – enabling the detection of more transcripts and genes from the same amount of sequencing.

Input Amount

Kit UHR RNA Input

Mapping (%)

Duplication (%)

Coverage uniformity

(CV)

Residual rRNA (%)

Strandedness (%)

# Unique transcripts

# Genes identified

KAPA Stranded RNA-Seq Kit with RiboErase

1000 ng 91.6 12.69 0.568 0.049 99.64 24,485,153 22,275

Illumina® TruSeq® Stranded Total RNA Library Prep Kit

1000 ng 92.6 21.77 0.663 0.156 99.95 23,047,496 21,782

KAPA Stranded RNA-Seq Kit with RiboErase

100 ng 90.3 26.77 0.618 0.064 99.50 19,824,805 22,376

Illumina TruSeq Stranded Total RNA Library Prep Kit

100 ng 92.6 34.04 0.639 0.126 99.94 19,425,920 21,898

NEBNext® Ultra Directional RNA Library Prep Kit with NEBNext rRNA Depletion Kit

100 ng 92.4 68.68 0.657 0.432 96.94 8,549,714 22,200

HBA1

[0 - 371]

p13.3 p13.2 p13.12 p12.3 p12.2 p12.1 p11.2 p11.1 q11.2 q12.1 q12.2 q13 q21 q22.1 q22.2 q23.1 q23.2 q24.1 q24.3

16

1,041 bp226,600 bp 226,800 bp 227,000 bp 227,200 bp 227,400 bp 227,600 bp

[0 - 103]

[0 - 253]

[0 - 166]

KA

PA10

0 ng

Illum

ina

100

ngN

EB10

0 ng

Tota

l R

NA

CoverageJunctions

CoverageJunctions

CoverageJunctions

CoverageJunctions

GC ContentGene

8,895 bp65,266,000 bp 65,267,000 bp 65,268,000 bp 65,269,000 bp 65,270,000 bp 65,271,000 bp 65,272,000 bp 65,273,000 bp

[0 - 9074]

[0 - 7904]

[0 - 11200]

[0 - 7299]

KA

PA10

0 ng

Illum

ina

100

ngN

EB10

0 ng

Tota

l R

NA

MALAT1

11

p15.4 p15.3 p15.1 p14.3 p14.1 p13 p12 p11.2 p11.11 q12.1 q12.3 q13.2 q13.4 q14.1 q14.2 q21 q22.1 q22.3 q23.1 q23.3 q24.1 q24.3 q25

GC ContentGene

Coverage

Junctions

Coverage

Junctions

Coverage

Junctions

Coverage

Junctions

Published by:

Roche Sequencing Solutions, Inc.4300 Hacienda DrivePleasanton, CA 94588

sequencing.roche.com

Data on file.For Research Use Only. Not for use in diagnostic procedures.KAPA is a trademark of Roche. All other product names and trademarks are the property of their respective owners. ©2017 Roche Sequencing Solutions, Inc. All rights reserved. SEQ100011 A038 02/17

Highly reproducible sequencing results• High correlation in gene expression levels between replicate samples, users and different test conditions

• Low sample-to-sample variation increases confidence in results

Ordering information

Roche Cat. No. KAPA Code Description Kit Size

07962282001 KK8483 KAPA Stranded RNA-Seq Kit with RiboErase (HRM) 24 rxn

07962304001 KK8484 KAPA Stranded RNA-Seq Kit with RiboErase (HRM) 96 rxn

High correlations of gene expression levels between different laboratories and RNA input amounts. This hierarchically clustered heatmap shows all correlations to be greater than 0.93, with the correlation between technical replicates produced in the same lab exceeding 0.98. Generally, all libraries from a single lab, including those produced from different input amounts, are clustered together. The data indicates that the KAPA Stranded RNA-Seq Kit with RiboErase delivers robust, reproducible, and unbiased gene expression data from a wide range of input amounts.

1.000

0.992

0.984

0.976

0.968

0.960

0.952

0.944

0.936

10 n

g

500

ng

100

ng -

S3

100

ng -

S1

100

ng -

S2

10 n

g

500

ng

100

ng -

S3

100

ng -

S1

100

ng -

S2

10 n

g

500

ng

100

ng -

S3

100

ng -

S4

100

ng -

S1

100

ng -

S2

10 ng500 ng

100 ng - S3100 ng - S1100 ng - S2

10 ng500 ng

100 ng - S3100 ng - S1100 ng - S2

10 ng500 ng

100 ng - S3100 ng - S4100 ng - S1100 ng - S2

Lab

1La

b 2

Lab

3

Lab 1 Lab 2 Lab 3

Log2 (replicate 2)

R2 = 0.9736

Log 2 (

repl

icat

e 1)

0 2 4 6 8 100

2

4

6

8

10

KAPA, 100 ng UHR

Log2 (replicate 2)

R2 = 0.9714Lo

g 2 (re

plic

ate

1)

0 2 4 6 8 100

2

4

6

8

10

Illumina, 100 ng UHR

Log2 (replicate 2)

R2 = 0.9598

Log 2 (

repl

icat

e 1)

0 2 4 6 8 100

2

4

6

8

10

NEB, 100 ng UHR

Excellent correlation of gene expression levels between replicate samples. Duplicate libraries were prepared from 100 ng UHR RNA, using the manufacturer's standard recommendations for each kit. The KAPA Stranded RNA-Seq Kit with RiboErase yielded a very high correlation (R2 = 0.9736) between replicates, and outperformed both competitors.