Automation of PacBio SMRTbell
10 kb Template Preparation on anAgilent NGS Workstation

Application NoteAuthors

Abstract

Nguyet Kong, Kao Thao, Whitney Ng,Automation of the PacBio 10 kb Template Preparation using the SMRTbell Templateand Bart C. Weimer

Prep kit for the sequencing of bacterial genomes on the Agilent Automated Liquid

100K Pathogen Genome Project

Handling Platform is described. Four bacterial genomes of differing GC content areDepartment of Population Health andused to prepare libraries without bias reading for next generation sequencing. TheReproduction

gDNA quality assessment was done using spectrophotometric and automated elec-

School of Veterinary Medicine

trophoresis for high molecular weight DNA with an Agilent 2200 TapeStation system

University of California, Daviswith the Agilent Genomic DNA ScreenTape assay. The sheared DNA was measured

Davis, CA, USAwith the Agilent Bioanalyzer system and the DNA 12000 Kit.

Kristi Spittle Kim, Jonas Korlach, andLuke Hickey
Pacific Biosciences
Menlo Park, CA 94025
Lenore Kelly and Stephen Lappin
Agilent Technologies, Inc.
Santa Clara, CA, USAIntroduction
Genome sequencing of microbial pathogens continue toreveal impactful insights into the complexities of infectious
disease agents, public health, human disease, and clinicaldiagnostics. Recent advancements in DNA sequencing technology allow whole genome sequencing to be accomplishedwith more cost-effective and high-throughput methods at an
unprecedented rate and sequence quality. In particular, theuse of next generation sequencing (NGS) methods has
increased productivity and revealed new information about
the structure and function of bacterial genomes, leading toincreasingly large data sets for analysis. Largely, NGS methods relying on short reads to produce draft genomes continue
to experience sequencing limitations due to GC content bias
and repeat regions that provide sufficient information to allow
closed genome assemblies. The PacBio RS II approach provides an alternative that quickly produces closed genomes byusing a single molecule, real-time (SMRT) DNA sequencing, atechnology that provides extra-long read lengths. This method
reduces the number of contigs and increases accuracy ofde novo assembly of bacterial whole genomes. The real-timetechnology is the only sequencing technology that allows
determination of the genomic DNA (gDNA) sequence, plasmid
DNA sequence, epigenetic modifications, and closed genome
assembly simultaneously. Due to these advantages, the 100K
Pathogen Genome Project (http://100kgenome.vetmed.
ucdavis.edu) selected SMRT technology to produce 1,000
closed genomes. Production of genomes on this scalerequired automation of the sequencing library construction.

Figure 1. The Agilent NGS Workstation. This workstation comprises thehighly reliable Agilent Bravo Automated Liquid Handling Platform,
Agilent Bravo accessories for heating, cooling, shaking, and magnetic bead manipulations. User intervention in multistep protocols
is minimized through the use of the BenchCel 4R Microplate
Handler and Labware MiniHub for labware storage and movement.

Materials and Methods
The SMRTbell Template Preparation kit from Pacific
Biosciences provides a simple, fast, and scalable solution for
producing a variety of insert-sized libraries that can be used ina broad range of NGS applications. The input gDNA must be
pure and of high molecular weight prior to fragmentation toachieve acceptable library size for optimal sequencing quality.
For this work, gDNA for PacBio SMRTbell 10 kb Library construction was extracted from selected bacterial isolates withincreasing GC content (Table 1). DNA was obtained with theQIAamp DNA Mini Kit (51306) using manufacturer’s instructions [1,2,3,4]. The gDNA quality assessment was done using
spectrophotometric and automated electrophoresis for highmolecular weight DNA Agilent 2200 TapeStation system with
the Agilent Genomic DNA ScreenTape assay, following
manufacturer instructions [5,6].

This application note describes the automation of the PacBio
10 kb Template Preparation using the SMRTbell Template PrepKit to provide rapid sequencing of bacterial genomes for the100K Pathogen Genome project, and obtain additional
genomic information linked to specific traits important tofood-associated bacterial pathogens. This solution consists ofthe industry’s most compact and versatile Agilent Liquid
Handling Platform (Figure 1), predeveloped protocols, andsolutions for labware management.

Table 1. Bacteria Used in This Study
ID no. Lab ID

2

ColorBacterium

GC content(%)

1BCW_5811

Campylobacter jejuni

302

BCW_3862

Listeria monocytogenes383BCW_5789

Vibrio fluvialis

414

BCW_4887

Salmonella enterica
serovar enteritidis

52

A streamlined workflow of the main steps to construct final
DNA libraries for sequencing was used:

After evaluation for gDNA size and quantity, fragmentation
was done using the Covaris g-TUBE device (520079) following
manufacturer instructions [7]. A sample loss between
20–50% was expected after fragmentation. The fragmented
gDNA was sized on an Agilent 2100 Bioanalyzer system withthe Agilent DNA 12000 Kit (part number 5067-1508). The
broad linear range of this separation gives both sizing andquantitation with minimal sample consumption [8,9]. The
fragmented DNA was quantified using a Life Technologies
Qubit 2.0 Fluorometer using a dsDNA HS assay, following
manufacturer protocol [10]. The sheared gDNA input into
library construction with the PacBio SMRTbell 10 kb Library
Preparation Kit was normalized for all samples between 1–5µg. If preparing libraries with DNA input amounts greater than
5 µg, all the reaction volumes can be scaled proportionally[11].

1. Fragment gDNA using a Covaris g-TUBE device.
2. Repair DNA damage and repair ends of fragmented DNA.
3. Purify the DNA.
4. Blunt-end ligate using blunt adapters.
5. Purify template for submission to a sequencer (Figure 2).
The workflow will take as little as 7 hours for post-shearing
cleanup and to construct DNA libraries for 96 samples readyfor submission using the NGS Workstation [12]. After library
construction, the sequencing facility anneals sequencing
primer and polymerase to the template before loading thelibrary onto the PacBio RS II.

Figure 2. SMRTbell Template Preparation Workflow for PacBio RS II system. This workflow is used to prepare libraries from fragmented and concentrated DNA
using Covaris g-TUBE and concentrated using the AMPure magnetic beads before following PacBio SMRTbell 10 kb Library Preparation procedures. If
preparing libraries with DNA input that are greater than 5 µg, scale the reaction volume proportionally.

3and reduce cost. The Post Shearing Cleanup Form and 10 kb
Library Prep Runset Dual SPRI Form, have many convenientfeatures, including various ways of tracking the progress
during a run, and intuitive manual control over instrumentcomponents to facilitate error recovery. The PacBio Library
Excel Workbook configures the number of plate sample
columns (1, 2, 3, 4, 6, or 12, that is, 8, 16, 24, 32, 48, or 96
samples), after which the interface displays the deck setup
and workstation setup for the protocol. The Excel workbook
is designed to guide reagent preparation and instrument
setup, and can be used to create a record of each experiment. Reagent volumes are calculated on the workbook,where lot numbers and notable information can be recorded.

In cooperation with Agilent Technologies, the 100K Pathogen
Genome Project and Pacific Biosciences automated and validated the PacBio SMRTbell 10kb Library Preparation process
on the Agilent NGS Workstation (G5522A) [12]. Two protocols, each with an easy-to-use graphic user interface, wereproduced (Figure 3) to enable quick scale-up of the protocol.
The VWorks Form is supplemented by an Excel Workbook,which facilitates reagent preparation and instruction setup,and can be used to keep a complete record of each experiment.
Each protocol has a unique deck layout displayed. The labware used in the protocol was selected for optimal performance to minimize dead volumes, conserve critical reagents,

BAC

Figure 3. VWorks protocol forms and Excel workbook for PacBio Library Preparation method provide an interactive, visual layout for the end user. A) Post
Shearing Cleanup Form. B) 10 kb Library Prep Runset Dual SPRI Form. C) PacBio Library Excel Workbook.

4

Results and Discussion

Workstation were used for QC. From 1 to 5 µg of sheared
gDNA from four different bacterial isolates of varying GC content (Figure 5), were used to construct libraries andsequenced on the PacBio RS II sequencer. Figure 6 shows theelectropherogram for the final DNA libraries for the organisms
listed in Table 1. The distribution of reads from each sample isplotted to determine the depth of coverage across thegenome.

High molecular weight gDNA used for library construction
was determined using an Agilent 2200 TapeStation system(Figure 4). Overlayed electropherograms and a virtual gel
image for the multiple sequencing libraries constructed using
PacBio 10 kb Library Preparation on the Agilent NGS12

341,400Sample intensity (FU)

1,200
1,000
800
600
400

48,5007,000

15,000

4,000

3,000

2,500

2,000

900

1,200
1,500

600200bp

Figure 4. Electropherogram and gel image of high molecular weight genomic DNA from an Agilent 2200 TapeStation system using the Genomic DNA ScreenTapeAssay. The electropherogram has been edited so that the lower marker is to the left of the y-axis, to focus on gDNA sample.

1234Figure 5. Representative electropherograms and virtual gels are used for visual inspection (generated with the Agilent 2100 Bioanalyzer system with the DNA
12000 Kit) of sheared bacterial genomic DNA with average shearing size for Campylobacter (green, 10 kb), Listeria (blue, 13.5 kb), Vibrio (aqua,11.6 kb), and Salmonella (red, 17 kb). Peaks near 35 are the lower marker internal standard for the DNA 12000 kit. A typical electropherogram using theAgilent Bioanalzyer 2100 DNA 12000 kit shows the lower marker at 35 seconds and the upper marker at 90 seconds. The sheared DNA and the uppermarker coelute together.

5

1

2

3

4

Figure 6. Representative electropherograms and virtual gel used for visual inspection (generated with the Agilent 2100 Bioanalyzer system with the DNA 12000
Kit) of DNA libraries sizes prepared for sequencing with the PacBio SMRTbell 10 kb Template Preparation Kit on the Agilent NGS Workstation. A typical electropherogram using the Agilent Bioanalzyer 2100 DNA 12000 kit shows the lower marker at 35 seconds and the upper marker at 90 seconds.
The DNA libraries and the upper marker coelutes with each other, the sharper peak is the upper marker. The average library sizes are: Campylobacter(green, 9.1 kb), Listeria (blue, 9.5 kb), Vibrio (aqua, 10 kb), and Salmonella (red, 15 kb).

Conclusion

Table 2 shows that microbial genomes with a different GCcontent had minimal bias from libraries made on the Agilent
NGS Workstation. The GC content observed by sequencing
matches closely with the known GC content on each genome
and no significant difference was observed. The results
demonstrate that the PacBio 10 kb Library Preparation
protocol is able to generate SMRTbell libraries from high complexity genomes with no significant coverage or GC bias in areproducible fashion on a NGS Workstation [13,14].

Automated protocols of PacBio 10 kb Library Preparation
generated similar technical performance to those produced
manually. With automation of the 10 kb SMRTbell library
preparation, up to 96 samples can be processed simultaneously. This is a marked improvement in throughput over themanual method without comprising the integrity of thesequence coverage or quality.

Acknowledgement

Table 2. Library Construction Metrics for Libraries Prepared with Each
Bacterium
Approx.
GCAvg.
Gram
genome size
content library
Bacterium
reaction (MB)(%)
size (kb)Campylobacter jejuni

Negative230

9.1

Listeria monocytogenes Positive

238

9.5

Vibrio fluvialis

Negative

10Salmonella enterica
serovar enteritidis

Negative541(2 chromosomes)
552

We gratefully acknowledge the technical assistance provided
by Carol Huang, Vivian Lee, Alan Truong, Lucy Cai, ChristinaKong, Alex Weimer, and Surene Foutouhi from the laboratoryof Dr. Bart Weimer in the School of Veterinary Medicine at theUniversity of California, Davis.

156

References

8. G2946-90004 the Agilent 2100 Bioanalyzer 2100 Expert
User’s Guide.

1. Qiagen QIAamp DNA Mini Kit:
http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/genomic-dna/qiaampdna-mini-kit#resourc

9. Agilent DNA 7500 and DNA 12000 Kit Guide (Rev. B),Agilent Technologies (p/n G2938-90024).
10. Qubit 2.0 Fluorometer User Manual, Life Technologies:
http://tools.lifetechnologies.com/content/sfs/manuals/mp32866.pdf

2. N. Kong, et al. Production and Analysis of High Molecular
Weight Genomic DNA for NGS Pipelines Using Agilent
DNA Extraction Kit. Agilent Technologies Application Notepublication number 5991-3722EN (p/n 200600).

11. PacBio Procedure & Checklist -Low-Input 10 kb Library
Preparation and Sequencing, http://www.pacificbiosciences.com/samplenet/ProcedureChecklistLowInput10kbLibraryPreparationandSequencingMagBeadStation.pdf

3. B. Ganesan, et al. Identification of the Leucine-to-2Methylbutyric Acid Catabolic Pathway of Lactococcus
lactis. Appl. Environ. Microbiol. 72:4264-73 (2006).

12. Agilent NGS Workstation (G5522A)

4. Y. Xie, et al. Expression profile of Lactococcus lactis ssp.
lactis IL1403 during environmental stress with a DNA
macroarray. Appl. Environ. Microbiol. 70:6738-6747 (2004).

13. D. Aird, et al. Analyzing and minimizing PCR amplification
bias in Illumina sequencing libraries. Genome Biol. 12: R18(2011).

5. Agilent 2200 TapeStation User Manual, Agilent
Technologies (p/n G2964-90001).

14. E.E. Schadt, et al. A window into third-generation
sequencing. Hum. Mol. Genet. 19: R227–240 (2010).

6. Agilent Genomic DNA ScreenTape System Quick Guide,
Agilent Technologies (p/n G2964-90040 rev.B).

For More Informatiom

7. Covaris User Manual g-TUBE, http://covarisinc.com/wpcontent/uploads/pn_010154.pdf

For more information on our products and services visit ourWebsite at www.agilent.com/chem.

7

www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.

© Agilent Technologies, Inc., 2014, 2015
Published in the USA
September 3, 2015
5991-4482EN