Application Note

High-Throughput NGS Library Prep for Plasmids and Amplicons withthe ExpressPlex™ Library Preparation Kit on the SPT Labtech firefly®
Introduction
In the synthetic biology domain, the ability to rapidly manufacture large numbers of new constructs andconfirm their sequences is of paramount importance. To leverage the ever-increasing capacity of sequencing
instruments while minimizing the sequencing cost-per-sample, users need to be able to multiplex larger
numbers of samples during every run. These dual requirements of reduced turnaround time and higher
multiplexing demand highly efficient NGS library preparation methods and reliable, easy-to-implement,automated liquid handling systems. Herein we describe the automation of seqWell’s ExpressPlex™ LibraryPreparation Kit on SPT Labtech’s firefly® liquid handler to create compact and cost-efficient methods forfast screening of synthetic constructs.

ExpressPlex Library Preparation Kit
The ExpressPlex Library Preparation Kit* is designed to be the fastest and easiest library prep kit available forcreating Illumina-compatible libraries from plasmid or amplicon DNA. Key features of ExpressPlex include:• 90-minute total time to final library, with only 30 minutes hands-on (for 96 samples)• Multiplexing up to 1,536 samples in a single run (6,144 barcodes expected early 2024)• Only 2 reagent additions, followed by a single cleanup step
• Auto-normalization of read counts and insert sizes• Up to 80% reduction in pipette tips required compared to other methods

ExpressPlex Workflow:* Patents Pending

For Research Use Only. Not for use in diagnostic procedures.

The ExpressPlex protocol requires only 2 pipetting
steps per sample prior to placing the full plate
onto the thermocycler. This enables the user toimmediately proceed to setting up the next
plate. Post-cycling, the user pools all samples
from the plate into a single tube and performs
one magnetic bead cleanup prior to quantifying
the library and loading onto the sequencer.
Technicians can easily prepare multiple sets oflibraries and begin sequencing in less than halfa day, and a high-throughput laboratory can easily
go from plasmids or amplicons to sequenced datain 24 hours. However, to take full advantage of themultiplexing available to ExpressPlex users, mostwill want to incorporate a robotic liquid handling
platform to maximize efficiency.

Application Note | ExpressPlex + SPT Labtech firefly

firefly Liquid Handler
The firefly platform is SPT Labtech’s newest, extremelycompact automated liquid handling platform. It measures
only 66cm (W) x 56cm (D) x 78cm (H), making it easy toplace in almost any laboratory that wants to improve
efficiency by automating manual workflows. Key featuresof firefly include:• 2 moving decks with a total of 16 deck positions• 2 liquid handling heads: an air-displacement pipetting
head and a non-contact positive-displacement dispensehead• An air-displacement pipetting head comprising 384
pipetting channels that can aspirate and dispense fromboth 96 and 384 well plates, depending on the formatof the tip array presented to the head
• A positive displacement head that can dispense up to
6 different reagents to 96 and 384 well plates on the deck
• A gripper to move plates• A loading position for the dispense head reagents• Optional application accessories for process modules
such as heaters or shakers
Visit sptlabtech.com/products/firefly for more information.

ExpressPlex + firefly
To assess the compatibility of seqWell’s library prep
solution and SPT’s robotic platform, we created a firefly
method that performs the library setup, which is followedby manual sealing of the plate and off-deck thermocycling. Post cycling, the plate is then moved back to
the firefly instrument for pooling, after which a simple
paramagnetic bead cleanup was performed. Final libraries
were sequenced on an Illumina MiSeq instrument anddata were evaluated for equivalence of read countacross all samples. For comparison purposes, the same
samples were prepared and sequenced manually.

For Research Use Only. Not for use in diagnostic procedures.

Application Note | ExpressPlex + SPT Labtech firefly

Materials and Methods for Automated and Manual Preparation ofExpressPlex Libraries
DNA Template
pUC19 plasmid DNA (New England Biolabs, Ipswich, MA) was diluted to 4 ng/uL and distributed across allwells of a 96-well plate.
Library Construction
Following the ExpressPlex Library Preparation Kit User Guide (seqWell, Beverly, MA), 4 uL of indexing reagent
was transferred from each well of an Indexing Reagent Plate to a Ready Reaction Mix Plate, 4 uL of DNA wastransferred to all wells of the Ready Reaction Mix Plate, the Ready Reaction Mix Plate was placed on athermocycler for fragmentation/indexing and amplification, and 10 uL from each well was ultimately pooled
into a 2 mL Eppendorf tube. Note: The firefly pools into a single column of a fresh plate, and the resulting
libraries are combined manually prior to cleanup.
Post-Pooling Cleanup
After pooling, libraries were purified in a conventional SPRI cleanup protocol using a 0.75 X ratio of seqWell’s
MAGwise paramagnetic beads to pool volume, typically 720 uL MAGwise to 960 uL pooled libraries.
Quantification for Concentration
Final purified libraries were quantified using the Qubit 4 Fluorometer and the Qubit 1X dsDNA HS Assay kit(ThermoFisher Scientific, Waltham, MA).
Electrophoresis
Fragment size distributions were characterized using the Agilent TapeStation 4200 and the High Sensitivity
D5000 ScreenTape System (Agilent Technologies, Santa Clara, CA). Key metrics provided by the TapeStation
Analysis Software include the average fragment size within the range of clusterable (sequenceable) fragment
sizes (400 – 1200 bp) and the fraction of the total which lies within the sequenceable range.
Quantification for Molarity
Libraries were diluted to 4 nM in Tris-HCl pH 8.
Final Preparation for Sequencing
Aliquots of each 4 nM library were pooled equi-volume and prepared for sequencing following the MiSeq
System Denature and Dilute Libraries Guide (Illumina, San Diego, CA). Denatured libraries were diluted to a
final loading concentration of 15 pM and sequenced on a MiSeq at 2 X 150 bp using the MiSeq Reagent Kit
v2 Micro 300 cycle kit (Illumina).

For Research Use Only. Not for use in diagnostic procedures.

Application Note | ExpressPlex + SPT Labtech firefly

ExpressPlex Library Preparation on the firefly
The deck layout for a single 96-well plate of ExpressPlex library preparation is shown below. For eachDNA sample plate to be sequenced, one Indexing plate, one plate of Ready Reaction Mix, and two tipboxes
are required, consuming a total of five deck positions. After library setup the plate is transferred to an offdeck thermocycler for fragmentation, indexing, and amplification. Space remains for an additional set of 96
to be prepared within the same run.

For Research Use Only. Not for use in diagnostic procedures.

Application Note | ExpressPlex + SPT Labtech firefly

ExpressPlex Library Preparation on the firefly
The deck layout for the sample pooling of a single 96-well plate is shown below. After thermocycling,
the Amplified Library Plate is placed on the firefly for pooling. For each plate of samples, a single column oftips and a column on an empty DNA plate is required. Up to four library plates can be pooled in a single run.

Liquid handling on the firefly is rapid - the total time to prepare 2 x 96-well plates of libraries (prior to thermalcycling) is less than 6 minutes, and the total time to pool 2 x 96-well plates of libraries is less than 10 minutes.

For Research Use Only. Not for use in diagnostic procedures.

Application Note | ExpressPlex + SPT Labtech firefly

Results
A total of 288 individual libraries were prepared across three separate 96-well plates on the SPT Labtech
firefly platform, as described above, and 96 libraries were prepared manually. The number of sequencing
reads per well, as a percent of the total reads recovered, reflects the consistency of the library preparation
across all libraries. As seen in Data Figure 1, the balance of sample reads is highly consistent, both interand intra-plate, for the automated and manual preparations, with coefficients of variation (CVs) of 9.9%, 9.5%,10.3%, and 8.8%.

Data Figure 1. Read Balances for three plates of pUC19 samples prepared and pooled on the firefly, with manual control data.
An ideal read balance would be 100%/96 wells, or 1.04% of total reads per well for all wells. The firefly produces read balances which
are close to the best attainable in actual practice, and highly comparable to read balances produced manually.

Visualized differently, the medians and distributions about the medians of the read-balance data for libraries
prepared using the firefly and libraries prepared manually are virtually indistinguishable (Data Figure 2).

Data Figure 2. Read balances for libraries prepared using the firefly and libraries prepared manually are practically indistinguishable

Highly similar read counts for pooled sample libraries are a result of the auto-normalization engineered into
the ExpressPlex Library Preparation Kit, and validates the ability to proceed directly from cleanup into final
sequencing preparation without the need to normalize sample libraries on a well-by-well basis.

For Research Use Only. Not for use in diagnostic procedures.

Application Note | ExpressPlex + SPT Labtech firefly

Discussion
Using the ExpressPlex Library Preparation Kit, the SPT Labtech firefly, and sufficient thermocyclers, a single
user can easily prepare 1,536 total libraries and take them through sequencing in approximately 24 hours.
One possible scenario divides the 16 plates comprising 1,536 samples into two cohorts of 8 plates apiece,and staggers the library preparation/thermocycling/pooling steps to maximize instrument and thermocycler
usage. To expedite the library preparation process, up to two 96-well plates can be set up for sequencing ina single firefly run, and up to four plates may be pooled in a firefly pooling run (additional programming may
be required).

Quick, reproducible sequencing of synthetic construct libraries can enable synthetic biology researchers to
rapidly determine if their sequences of interest are present. With the strategy described in this application note,high-throughput laboratories can efficiently screen large numbers of constructs and focus on the importantdiscovery aspects of their work.

For Research Use Only. Not for use in diagnostic procedures.

Application Note | ExpressPlex + SPT Labtech fireflyConclusions• ExpressPlex libraries created on the SPT Labtech firefly are highly consistent, maintaining highly balanced
read counts without normalization.
• Users can prepare and sequence 1,536 or more samples in 24 hours, which enables speedy discovery via
rapid data turnaround.
• Through high-throughput screening of synthetic constructs, researchers can increase their rates of discovery,
thus allowing them to focus on more important aspects of their work.

The following scientists are responsible for the work shown in this application note:
John Palys (seqWell), Anita Pearson (SPT), Ian Whitmore (SPT), and Huw Rees (SPT)

Would you like more information about ExpressPlex, firefly, or associatedmethods? Contact seqWell or SPT Labtech, respectively, at:
Visit: seqwell.com/contact

Unless otherwise stated in references, presented data on file.
For Research Use Only. Not for use in diagnostic procedures.
All product names and trademarks are the property of theirrespective owners. © 2023 seqWell Inc. All rights reserved.

Visit: sptlabtech.com/contact