Automation of NuGEN Ovation
RNA-Seq System V2 on the Agilent
NGS Bravo

Application NoteAuthors

Introduction

Lisa KnappThe Ovation RNA-Seq System V2 from NuGEN Technologies provides a fast andsimple method for preparing ampliﬁed cDNA from total RNA for RNA-Seq applications,with enhanced transcript coverage and uniform distribution of sequencing reads.
Ampliﬁcation is initiated at the 3’ end as well as randomly throughout the wholetranscriptome in the sample. This feature makes the Ovation RNA-Seq System V2 ideal
for ampliﬁcation prior to Next Generation Sequencing, as it allows ampliﬁcation of bothmRNA and non-polyadenylated transcripts.

Agilent Technologies, Inc.
1362 Kirklees DrCarmel, IN 46032
Lin Z Pham
NuGEN Technologies, Inc.
201 Industrial Road, Suite 310
San Carlos, CA 94070

The Ovation RNA-Seq System V2 is enabled by Ribo-SPIA technology, a rapid, simple,and sensitive RNA ampliﬁcation process developed by NuGEN. Using Ribo-SPIA
technology and starting with as little as 500 pg total RNA, µg quantities of cDNA can beprepared in approximately 4.5 hours.
The Ovation RNA-Seq System V2 (NuGEN part number 7102) provides optimized reagent
mixes and a protocol to process eight or 32 total RNA samples, and is also available as
an automation solution for processing 96 samples.

Automated protocols are now available
for the Ovation RNA-Seq V2 workﬂow on
Agilent NGS Bravo (part number G5541A)(Figure 1).

Experimental Setup
The NuGEN Ovation RNA-Seq System
V2 form-based protocols provide aninteractive, visual layout for the enduser. The underlying VWorks protocols
have been designed to allow laboratory
personnel to use the same protocol
to run one to 12 columns of samples.
Preparation of double-stranded cDNAfrom total RNA for 96 samples can beaccomplished in less than 5 hours. Eachprotocol has a unique deck layout, whichis shown in Figure 2. This is also used tostart the protocol and select options suchas the number of columns of samples toprocess.
The labware used in the protocol wasselected for optimal performance while
being cognizant of minimizing deadvolumes when reagent conservationis critical and ease of setup for thosereagents where cost is negligible. The
Current Tip State selection allows partial
boxes of tips to be used to minimize tipwaste.

Figure 1. Agilent Bravo platform for NGS.

Figure 2. VWorks protocol form.

2

Links are provided to a spreadsheet,
which calculates volumes for all mastermixes (Figure 3) and has space provided
to enter lot number information or
any other information which may berequired for documentation within thelab.

Results and Discussion
Two nanograms of Universal Human
Reference total RNA (MAQC A - partnumber 740000) and Brain total RNA(MAQC B) were used to generate cDNA
using the Ovation RNA-Seq System V2(part number 7102) on Agilent NGS Bravo.
One hundred nanograms of the resulting
cDNA was used to produce sequencing
libraries using the NuGEN Ovation
Ultralow Library System on the Agilent
NGS Bravo.
Table 1 shows the sequencing alignment
metrics with the expected level of
uniquely mapped reads, low levels ofrRNA reads, and good detection of
RNA-Seq transcripts, as compared to thecontrol data from manual preps.

Conclusion
Automated protocols of Ovation
RNA-Seq System V2 generated similar
technical performance as compared tothe manual process. With automation,up to 96 samples can be processedsimultaneously. This is a marked
improvement in throughput over themanual method without comprising
the integrity of the experiment. TheOvation RNA-Seq System V2 is available
from NuGEN in a 96 reaction size
(part number 7102-A01).

Figure 3. Volume tables and data sheet.

Table 1. Sequencing alignment metrics.
SampleMethodTotal reads% Mappedreads% Uniquereads% rRNAreads# Ref-Seq
genes

Brain

Bravo

14,895,950

86

61.89

38.30

15,970UHR

Bravo

13,238,807

87

56.59

35.40

16,037

UHR_rep1

Manual13,531,223

66

40.33

36.10

15,544UHR_rep2

Manual

14,051,431

67

41.05

37.10

15,5003

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For Research Use Only. Not for use in diagnostic
procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2013, 2016
Published in the USA, March 10, 2016
5991-3598EN