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

Authors

Lisa Knapp

Agilent Technologies, Inc.

1362 Kirklees Dr

Carmel, IN 46032

Lin Z Pham

NuGEN Technologies, Inc.

201 Industrial Road, Suite 310

San Carlos, CA 94070

Introduction

The Ovation RNA-Seq System V2 from NuGEN Technologies provides a fast and 

simple 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 whole 

transcriptome 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 both 

mRNA and non-polyadenylated transcripts. 

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 be 

prepared 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 an 

interactive, visual layout for the end 

user. 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 cDNA 

from total RNA for 96 samples can be 

accomplished in less than 5 hours. Each 

protocol has a unique deck layout, which 

is shown in Figure 2. This is also used to 

start the protocol and select options such 

as the number of columns of samples to 

process. 

The labware used in the protocol was 

selected for optimal performance while 

being cognizant of minimizing dead 

volumes when reagent conservation 

is critical and ease of setup for those 

reagents where cost is negligible. The 

Current Tip State selection allows partial 

boxes of tips to be used to minimize tip 

waste. 

Links are provided to a spreadsheet, 

which calculates volumes for all master 

mixes (Figure 3) and has space provided 

to enter lot number information or 

any other information which may be 

required for documentation within the 

lab.

Results and Discussion

Two nanograms of Universal Human 

Reference total RNA (MAQC A - part 

number 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 of 

rRNA reads, and good detection of 

RNA-Seq transcripts, as compared to the 

control data from manual preps. 

Conclusion

Automated protocols of Ovation 

RNA-Seq System V2 generated similar 

technical performance as compared to 

the manual process. With automation, 

up to 96 samples can be processed 

simultaneously. This is a marked 

improvement in throughput over the 

manual method without comprising 

the integrity of the experiment. The 

Ovation RNA-Seq System V2 is available 

from NuGEN in a 96 reaction size 

(part number 7102-A01).