Automated NGS Library Prep
Next Generation Sequencing (NGS) library preparation is a critical step in the sequencing workflow, involving the processing of DNA or RNA samples into a format compatible with NGS platforms. Myra functions as an automated NGS library prep workstation, offering reliable performance for this essential process.
This also includes bead clean up, which can be used for PC product isolation or fragment purification for applications outside of NGS. The Myra NGS liquid handler helps streamline these tasks for efficiency and accuracy.
Capable of handling up to 24 libraries, Myra incorporates a 96 Well Magnetic Station for bead clean-up steps, with off-deck heating to reduce robotic downtime. The workflow is split into manageable runs, with safe stopping points after the various library preparation steps, including bead clean up. This makes Myra ideal for automated NGS library prep and related workflows.
In NGS, repeatability and reproducibility are paramount. The Myra liquid handling system can successfully generate high-quality DNA libraries, with performance comparable to manual methods. Sequencing runs show high Q30 scores and consistent barcode balance. Sequencing coverage exhibits uniformity across the prepared libraries, resulting in low library variability and reliable performance. Myra supports precise NGS sample preparation liquid handling, reducing manual labor while delivering high-quality sequencing results.
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Case Study: Illumina DNA Prep Kit
We validated up to 24 samples using the Illumina DNA Prep kit. Libraries displayed low variability across replicates, even barcode balance, and high coverage of sequence alignments. This highlights the value of Myra as an automated NGS library prep workstation for large-scale applications.
Experimental Setup
To demonstrate capability, 8-sample and 24-sample plates of 150 ng of input DNA (48 kB genome Lambda DNA and Human Genomic DNA) were prepared on the Myra Liquid Handling System. Manual preparation of an 8-sample plate of 150 ng of input DNA (48 kB genome Lambda DNA) was also performed as a comparison method. The quality of these libraries was checked with BiOptic Qsep1-Plus bio-fragment analyzer (Figure 1). The libraries were quantified using NEBNext Library Quant Kit for Illumina with reaction setup on the Myra Liquid Handling System and amplification on the Mic qPCR Cycler. Libraries were simultaneously normalized and pooled to 2 M using the Myra Liquid Handling System, demonstrating reliable liquid handling NGS sample preparation. Sequencing was performed on a 2×150 bp flow cell on an iSeq 100 System for whole genome sequencing. Q30 and percentage PF were used to show the quality of the libraries. Sequencing data was demultiplexed using Illumina’s BaseSpace Sequence Hub, and coverage maps of the Lambda genome were analyzed using Geneious Prime 2022.2.2.
Results
We were able to successfully generate high-quality 8-plex and 24-plex DNA libraries on the Myra, with comparative performance to the manual method (Figure 2). Fragment analyzer results show consistent fragment sizes across all 24 DNA libraries (Figure 1). The quality of the sequencing runs was comparable with a Q30 score > 90% for all methods. Barcode balance was also accurate, with variability less than the manual method. Sequencing coverage of the Lambda genome showed consistent uniformity across 8-plex and 24-plex DNA prep libraries constructed on the Myra. The sequence data showed low sample variability and reliable uniformity for libraries prepared using Illumina DNA Prep, reinforcing the benefits of using an automated NGS library prep workstation.
Additional Accessories
The following accessories are recommended for achieving library prep on the Myra liquid handling system.
Myra 96 Well Magnetic Station
MYRA-MAGS96
Designed to provide automated magnet control for up to 96 x 0.2 mL tubes, including strips of 8, and 96 tapered well plates (including fully skirted, unskirted and semi-skirted plates). Magnets are applied to groups of 24 wells at a time. Made from aluminium.
Myra 96 Well Cooling Block
MYRA-COOL96
Designed to provide passive cooling for 96 x 0.2 mL tubes including strips of 8, and 96 tapered well plates (including fully skirted, unskirted and semi-skirted plates).
Frequently Asked Questions
What is NGS library preparation and why is it critical?
NGS library preparation converts DNA or RNA samples into a form that can be sequenced by NGS platforms. It ensures that each sample is ready for accurate sequencing with reliable results.
How does Myra automate the NGS library prep workflow?
Myra functions as an automated NGS library prep workstation, streamlining key steps through precise liquid handling for reagent dispensing, bead cleanup, quantification, normalisation and pooling. Its integrated 96 Well Magnetic Station enables accurate magnetic bead-based cleanups, while programmable scripts manage pipetting volumes and timing with high precision. This reduces variability, ensures consistent reagent mixing and supports reliable barcode balance and uniform sequencing coverage across all libraries.
What are the main challenges in manual NGS library preparation that Myra addresses?
Manual prep is labor-intensive and prone to variability due to inconsistent pipetting and timing. Myra solves this by:
- Automating liquid handling with high precision
- Reducing human error
- Providing consistent timing for bead clean-up and reagent addition
- Enhancing reproducibility and sample-to-sample consistency
Is Myra’s library prep performance comparable to manual methods?
Yes, Myra delivers high-quality DNA libraries with consistent performance that matches manual preparation. It ensures accuracy across all steps of the NGS sample preparation liquid handling process.
Can Myra handle cleanup and normalization in the same workflow?
Where Can I Learn More?