Automation of NuGEN Ovation
Ultralow Library Systems on anAgilent NGS Bravo

Application NoteAuthors

Introduction

Lisa KnappWith the reduction in cost and increase in throughput, genetic sequencing is going
beyond traditional total genome sequencing to multiple new and exciting life science
applications. As such, laboratories have to face the challenge of consistently runninga large number of samples through the entire NGS process, starting with sample
preparation. For large volume studies, automation of the next-generation sequencing(NGS) sample preparation process including DNA/RNA isolation, cDNA preparation,library construction, multiple puriﬁcation, quality control steps, and potentially target
enrichment steps, offers signiﬁcant advantages in terms of manual labor reduction andelimination of operator induced errors and variability. Agilent NGS automation solutions
streamline sample preparation for a variety of applications to provide excellent reliability
and reproducibility. The solution consists of the industry’s most compact and versatile
Bravo Automated Liquid Handling Platform (Figure 1), predeveloped protocols andoptional solutions for labware management.

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 Ultralow Library Systems from NuGEN Technologies provide a simple, fastand scalable solution for producing libraries that can be used in a broad range of NGS
applications starting with 1 to 100 ng of DNA. The methods used for fragmentation
and adaptor ligation provide for low bias libraries suitable for RNA-Seq, Digital GeneExpression (DGE), genomic DNA sequencing, target capture, amplicon sequencing,
ChIP-Seq, and more.

As shown in Figure 2, the streamlined
workﬂow consists of four main steps:
1.

Fragmentation of either genomic
DNA or double-stranded cDNA

2.

End repair to generate blunt ends

3.

Adaptor ligation for multiplexing or
no multiplexing

4.

PCR ampliﬁcation to produce theﬁnal library

The entire workﬂow, including
fragmentation, can be completed inas little as four hours and yields DNA
libraries ready for cluster formationand either single read or paired-end
sequencing.

Figure 1. Agilent Bravo platform for NGS.

Input DNA

Fragment

5’3’

End repair5’ P

3’P

Add adaptors and ligate
Bead puriﬁcation
Fill in and PCR

Bead puriﬁcation
Cluster formationand sequencing

AATCGGATAGGTAGGAT...
TCTCGATGCAAGTGATC...
GTAGCAAAATCCTGAGA...

Figure 2. The Ovation Ultralow Library Systems Workﬂow.

2

Automated protocols are now available
for the NuGEN Ovation Ultralow Library
Systems on Agilent NGS Bravo (partnumber G5541A). An easy to use graphic
user interface (Figure 3) enables a quick
scale up of the protocols for any lab.

Experimental Setup
The NuGEN Ovation Ultralow Library
Systems 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.
Library construction for 96 samples takes
approximately 4 hours. Each protocol hasa unique deck layout which is shown in
Figure 3. The labware used in the protocol
was selected for optimal performance
while being cognizant of minimizing deadvolumes when reagent conservationis critical, and ease of setup for thosereagents where cost is negligible. TheCurrent Tip State selection allows partial
boxes of tips to be used to minimize tipwaste.

Figure 3. VWorks protocol form.

Links are provided to a spreadsheet
which calculates volumes for all mastermixes (Figure 4) and has space provided
to enter lot number information or any
other information which may be requiredfor documentation within the lab.

Figure 4. Volume tables and data sheet.

3

Results and Discussion
Multiple sequencing libraries wereconstructed using Ovation Ultralow
Library System on a NGS Bravo with
1.0 ng of sheared genomic DNA fromE. Coli (GC: 51 %), Staphylococcus
aureus (low GC: 33 %), and Rhodobacter
sphaeroides (high GC: 69 %), respectively.
Triplicate sequencing libraries for eachbacteria were sequenced on an Illumina
sequencer. The distribution of reads fromeach sample is plotted to determine thedepth of coverage across the genomes.
Sequencing data from manual library
prep were also plotted for comparison.
Figures 5A, 5B, and 5C show thecoverage plots for E. coli, Staphylococcus
aureus, and Rhodobacter sphaeroides,
respectively. The grey track represents
the theoretical distribution, and the othertracks are from the experimental data
generated from either the Bravo library
prep or the manual prep.

×105876
Manual_rep1
Manual_rep2
Manual_rep3
Simulated
Bravo_rep1
Bravo_rep2
Bravo_rep3

5432
1005101520253035

Figure 5A. Coverage plot from sequencing libraries with 1 ng of E coli genomic DNA.
×105
3.0

2.5

Manual_rep1

2.0

Manual_rep2
Manual_rep3
1.5

Bravo_rep1
Bravo_rep2
Bravo_rep3

1.0

Simulated
0.5005101520253035404550

Figure 5B. Coverage plot from sequencing libraries with 1 ng of Staphylococcus aureus genomic DNA.
×105
65
Manual_rep1

4

Manual_rep2
Manual_rep3

3

Bravo_rep1
Bravo_rep22

Bravo_rep3
Simulated

1005101520253035

Figure 5C. Coverage plot from sequencing libraries with 1 ng of Rhodobacter sphaeroides genomic DNA.

4

Shown in Table 1, minimal bias wasobserved with microbial genomes having
extreme GC content, 33 % (S. aureus),and 69 % (R. sphaeroides), as well as
E. coli genome (~51 % GC) from libraries
made both manually or on the NGSBravo. The GC content observed bysequencing matches closely with theknown GC content of each genome, andno signiﬁcant difference was observed
between the manual and Barvo library
prep.

Table 1. Sequencing alignment metrics with a range of GC content.
Species

Library prep
methodTotal reads

Alignedreads (%)

Known GC
content (%)

Observed GC
content (%)

E. coliManual1,000,000

98.8

50.8

50.6

E. coli

Bravo

1,000,000

99.4

50.8

50.4

S. aureus

Manual1,000,000

99.2

32.8

36.4

S. aureus

Bravo

1,000,000

99.3

32.8

35.1R. Sphaeroides

Manual

1,500,000

99.0

68.5

67.5

R. Sphaeroides

Bravo

1,500,000

99.1

68.5

67.1

Taken together, these results demonstrate
Ovation Ultralow Library Systems
generate high complexity libraries withno signiﬁcant coverage or GC bias ina reproducible fashion on Agilent NGS
Bravo.

Conclusion
Automated protocols of Ovation Ultralow
DR Multiplex Systems generated similar
technical performance as compared tothe manual process. With automation
and NuGEN barcode designs, up
to 96 samples can be processed
simultaneously. This is a marked
improvement in throughput over manual
method without comprising the integrityof the experiment. The Ovation Ultralow
DR Mutiplex System is also available
from NuGEN in a 96 reaction size
(part number 0329-96)

5www.agilent.com/chem
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
5991-3599EN