With the rapid growth of Next Generation Sequencing (NGS), having a robust, automation-friendly library preparation solution is a minimum requirement for handling precious samples across many applications.
The automation of the library preparation process offers multiple advantages, including increased throughput, reduced performance variability, and potential error elimination in sample tracking. Nevertheless, transitioning from manual workflows to automation can often appear daunting and time-consuming.
This article discusses TGen North’s process of assessing various library preparation solutions and automation systems. The aim is to transition from the current manual library preparation method to an automated workflow, utilizing SPT Labtech’s firefly® in combination with the Watchmaker DNA Library Prep Kit with Fragmentation.
Introduction
TGen North, located in Flagstaff, Arizona, is home to TGen’s Pathogen and Microbiome Division. The HIVE Laboratory at TGen North plays a crucial role in surveillance programs for multiple pathogens, aiding the state of Arizona in informing the public and assisting underserved communities.
Whole genome sequencing is used to monitor emerging outbreaks and facilitate informed public health responses. This group manages high throughput sample processing, including NGS, covering specimen accession, extraction, QC, sequencing, and data analysis.
With the critical need for accurate pathogen surveillance, manual library preparation processes have posed challenges, including monotonous workflows with an increased risk of potential human errors and inconsistencies between lab personnel.
In pursuit of enhanced efficiency and data quality, this article will discuss the transition to automated library preparation processes.
The transition to automation was ultimately enabled by first identifying areas for improvement in the overall workflow and then determining evaluation criteria for both sample prep solutions and automation platforms to be considered.
Comparison experiments were then performed to identify the most robust sample prep solution and last working with the selected automation vendor, SPT Labtech, to automate the library prep workflow of choice, the Watchmaker DNA Library Prep Kit with Fragmentation.
The subsequent sections will delve into this transition towards automation and its potential to transform NGS workflows, ensuring more efficient and precise analyses with reliable and reproducible outcomes.
Manual Processing – Identifying Areas for Improvement
Automating library preparation can reduce the number of error-prone steps and the time scientists in the laboratory need. This enables them to focus on more intricate tasks.
Image Credit: SPT Labtech
Time-Consuming and Error Prone Steps
Normalization
- Arranging samples in an array
- Normalizing samples with different dilution requirements
Library Construction
- Purifying beads and selecting the appropriate size
- Variability in liquid handling among scientists
- Transferring and tracking samples
Library Pooling
- Missed samples and pipetting errors
- Uneven sequencing results
Why Automate?
- Speed and reliability
- Prevention of cross-contamination
- Reduction of human errors in indexing, pooling, and sample tracking
- Tracking of protocol versions
- Consistency in the library yields
- Reliable sequencing results, which lower sequencing costs
Several automated liquid handling systems from different vendors were considered. Ultimately, TGen North chose firefly for the following reasons:
- Cost: It costs roughly half as much as other systems
- Lab footprint: firefly’s z-height design allows it to fit on a standard benchtop
- Intuitive software: It has intuitive, cloud-enabled software
- Support: SPT Labtech’s field application scientists and field service engineers are responsive and helpful in developing new protocols
- Flexibility: The system has 16 deck positions, a non-contact dispenser, and dual-core capability for handling 384, 96, and strip tips, along with an optional integrated shaker and plate thermal module
- Efficiency: It requires significantly less reagent dead volume for automating library prep workflows due to the integrated non-contact dispenser
Determining the Appropriate DNA Library Prep Kit to Scale
Seven DNA library prep kits were compared using the same eight independent DNA isolates from GM12878 cells, following the Chemagic Cell Pellet protocol from Revvity to eliminate process variability.
In addition to processing positive controls, negative extraction controls (NEC) and non-template controls (NTC) were also processed for each kit, using the input quantities recommended by each kit.
Evaluation Criteria for Whole-Genome Library Preparation
Quality Fragmentation Enzyme
- Consistent results
- Avoid the need for double-sided selection
- Ligation and conversion efficiency
- Input quantity range
- Ease of protocol
- Time requirements
- Support
- Technical, sales, automation
- Minimal sequencing artifacts and bias
- GC, start-bias
Figure 1. Conversion efficiency comparison. Calculation assumes no sample loss through process. (((qPCR Molarity [pM] / 1x109) x (size [bp] x 660 x volume [uL]) / (input DNA [ng] )) x 100. Image Credit: SPT Labtech
Figure 2. D5000 TS assay for Watchmaker DNA Library Kit with Fragmentation. No additional size selection required. Even sizing and yield across samples (pink), Clean negative controls (teal), Positive control (dark grey), Ladder (light grey). A conversion efficiency for Watchmaker WGS with 4 cycles of PCR amplification and 100 ng of DNA was 70-80% (likely higher, post ligation product is diluted 2x). Conversion efficiency was calculated using Tapestation data as follows: (((Library [ng/uL] x volume [uL]) / (1.65^#PCR cycles)) / Input DNA [ng]) x100. Image Credit: SPT Labtech
Figure 3. Virtual Gel Image of Watchmaker final libraries from 8 independent DNA isolates (F6-E7), ladder (L4), positive control (F7), negative controls (G7-H7). Image Credit: SPT Labtech
The Watchmaker DNA Library Prep Kit with Fragmentation comes equipped with a sturdy enzymatic fragmentation module that allows for adjustable and consistent outcomes (see Figure 2), even across various inputs, without the need for extra size selection steps.
Among the seven kits that were tested, the Watchmaker DNA Library Prep Kit with Fragmentation demonstrated the highest conversion rates (refer to Figure 1) while maintaining a simplified and automation-friendly workflow (as shown in Figure 4), which is compatible with PCR-free options.
This kit also includes sufficient extras for automation, and Watchmaker boasts a knowledgeable scientific support team ready to assist as required.
Figure 4. Watchmaker DNA Library Prep Kit with Fragmentation workflow overview. Image Credit: SPT Labtech
Conclusion
In conclusion, the adoption of automated library preparation using the Watchmaker DNA Library Prep Kit with Fragmentation on firefly has markedly enhanced TGen North’s pathogen surveillance workflows.
The robust enzymatic fragmentation module, excellent conversion rates, and streamlined automation-friendly workflow provided by the Watchmaker kit have played a crucial role in achieving improved efficiency and data quality.
The chosen firefly automation system offers cost-effectiveness, space efficiency, user-friendly software, and outstanding support from SPT Labtech’s scientific team.
With increased flexibility and reduced reagent waste, automated library preparation has empowered TGen North to handle valuable samples more effectively, ensuring timely pathogen surveillance and freeing up resources for more intricate tasks in the laboratory.
About SPT Labtech
SPT Labtech designs and manufactures robust, reliable and easy-to-use solutions for life science. We enable life scientists through collaboration, deep application knowledge, and leading engineering to accelerate research and make a difference together. We offer a portfolio of products within sample management, liquid handling, and multiplexed detection that minimize assay volumes, reduce material handling costs and put the discovery tools back in the hands of the scientist.
At the Heart of What We Do
Many of our innovations have been born out of the desire to create solutions to existing customer problems; and it’s this ethos that drives SPT Labtech’s R&D efforts. Our strengths come from the trust our customers have with us to develop truly unique, automated technologies to meet their needs. We combine cutting edge science with first-rate engineering to put customers at the heart of everything we do.
A Problem-Solving State of Mind
The substantial breadth of expertise within our company enables us to be involved in the full life cycle of our products from the initial design concept, mechanical and software engineering and prototyping, to final manufacture and sale. These qualities allow us to offer the best possible technical and mechanical support to all the equipment that we supply, hence maintaining excellent client relationships.
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