Title

Democratizing Automated Sample Prep – Low Cost Automation of Next Gen
Sequencing Library Prep for Moderate Throughput Laboratories
Title
www.lucigen.com
Curtis
Knox*, Svetlana Jasinovica*, Michael Lodes*, Sally Floyd*, David Mead*, Kevin Barrett**, Tristan Berto**, Seth Hanson**
Authors
*Lucigen
Corporation , Middleton, WI, USA ** Gilson, Inc., Middleton, WI, USA
Location
ABSTRACT
With the advent of more affordable “personal” next
generation sequencing instruments from major
platform providers, NGS applications are moving
outside of core sequencing labs to enable a variety
of projects. As the number of samples for
sequencing increases, there is a need for library
prep automation that matches throughput of the
instruments while not requiring an investment equal
to the sequencer itself. We are developing a
combination of new NGS sample prep chemistry and
a small automated platform that reduces time to final
prepared library and increases efficiency and
consistency. The DNA sample prep chemistry
combines typical end-repair and A-tailing steps into
one master mix step with buffers directly compatible
with downstream ligation steps, eliminating the need
for multiple cleanup steps throughout the process.
The chemistry has also been optimized to drive
higher A-tailing efficiencies, which reduces chimera
formation from blunt fragments and loss of fragments
tailed with nucleotides other than the necessary “A”.
These chemistries were designed to be easily
automatable, and subsequent development led to
the design of a compact liquid handling platform to
perform these tasks. The instrument described in
this presentation allows numerous DNA libraries to
be prepared simultaneously, including incorporation
of barcoded adapters for multiplex PCR and
sequencing. In contrast to other small, dedicated
systems, the instrument is also open and
programmable, meaning users can choose to utilize
the platform for other applications. In this poster, we
will discuss the feasibility of automating the
chemistry on the instrument and the utility of an
inexpensive NGS sample prep system for
laboratories with a range of sample throughput
needs.
NxSeq® Sample Prep Technology
The NxSeq DNA Sample Prep kit was developed to:
• Reduce time to complete NGS sample prep.
• Improve A-tailing and downstream ligation of
fragments to adapters.
An optimized master mix of enzymes and buffers
enables end-repair and A-tailing of DNA fragments in
one tube without the need for buffer exchange or
sample cleanup. The result is a manual system for
NGS sample prep from sheared DNA that cuts
traditional workflow by up to 50% (2 hours) from the
most commonly used method. In addition, hands-on
time is reduced by up to 75% (see figure 1).
NxSeq® DNA Prep Kit Validation
Previously, we validated the performance of the
NxSeq DNA Sample Prep kits by shearing DH10B E.
coli genomic DNA and preparing identical samples
with the TrueSeq® Sample prep kit v2, The Roche
454 Sample prep kit, and the NEBNext ® DNA
Sample Prep Master Mix Set 1. All samples were
verified for target size range on an Agilent
Bioanalyzer and then run on an Illumina Genome
Analyzer IIx using barcoded adapters (see figures 2
and 3).
Fig. 5 – Protocol builder software for the PIPETMAX.
NxSeq/PIPETMAX® System
Figure 2. Bioanalizer trace comparison of DH10B Genomic
DNA libraries made with NxSeq, NEB, and Roche prep kits.
Equivalent performance was obtained for all kits.
The goal of the Lucigen/Gilson collaboration is to
develop a turn-key automated method for NGS
sample prep utilizing sheared DNA as the input and
returning adapter-ligated DNA size selected to
appropriate platform-specific ranges. The ability to
select individual barcode adapters for ligation to
multiple samples and minimal manual interventions
are also aims of the project.
Table 1: Capacities of the NxSeq/PIPETMAX
Method
Figure 3. Coverage comparison of DH10B Genomic DNA
libraries. Plots indicate depth of coverage achieved in a
moving 100 base read window. Equivalent performance was
obtained for all kits.
PIPETMAX®
Platform
Gilson, Inc . is a recognized leader in manual and
automated liquid handling solutions. The
PIPETMAX is an easy-to-use, personalized,
automated liquid handling platform designed for turnkey research application solutions. The system
features exchangeable pipetting heads with a range
of volume handling from 1 – 1,000 µL. Two heads
can be used at one time and are easily switched out
by hand. The tray base is customizable for different
plate usage depending on application, and the
moving XYZ system can reliably target 384-well
plates. The instrument is fully enclosed to protect
samples, but the enclosure is removable for use
within a fume hood (see figure 4). Easy to use
software is included for protocol configuration (see
figure 5).
Time to run 8 samples
~3 hours
Samples per run – single
adapter
1-96
Samples per run –
barcoded adapters
1-24
Manual intervention steps
1
Automated System Optimization
To optimize performance of the automated method,
we will prepare NGS samples from DH10B genomic
DNA using NxSeq chemistry both manually and on
the PIPETMAX.
For the manual preparation, all steps will be
performed as described in the product manual. For
the automated version, all reagents will be placed on
the deck of the platform, with the method running
without interruption except for manually moving a
single plate to and from a thermal cycler for the
heated incubation steps. Both ligation to a barcoded
adapter, and size selection (200-500 bp target
range) will be performed on-deck with size selection
using a modified AMPure XP bead protocol.
Optimization experiments will include:
• Range of wash drying times to facilitate removal of
alcohol.
• Pipetting speeds and volumes to eliminate bead
carryover.
• Minimizing time to final sample and tip usage.
Conclusions
The PIPETMAX instrument, combined with NxSeq
DNA Prep Kit chemistry, is capable of preparing DNA
libraries for NGS applications with selectable size
ranges specific to platform needs. Optimization
experiments are ongoing and will result in a system
that will be suitable for hands-free, barcoded library
preparation on a compact, cost effective, liquid
handling platform.
We anticipate the fully validated method to be
available through Gilson as part of the PIPETMAX
package in Spring, 2013.
Fig. 1 – Comparison of total time and hands-on time
required for sample prep between NxSeq and common
commercially available kits.
Fig. 4 – the PIPETMAX instrument.