Exploring DNA Library Preparation Miniaturization for Illumina Sequencing Technology

Advancements in next-generation sequencing have led to significant increases in throughput and reductions in associated costs. As a result, the process of library preparation has become a more substantial financial and time constraint for high-throughput sequencing core facilities.

The utilization of liquid handling robots, specifically the mosquito HV system, has helped alleviate some of these bottlenecks. This article outlines the successful implementation of the mosquito HV system in preparing DNA libraries for Illumina sequencing, reducing the volume to one-tenth of the original manual protocol.

Key Benefits

mosquito HV genomics, a fully open liquid handler:

  • Provides low-cost DNA library preparation through miniaturization of reagent volumes
  • Increases reproducibility and data quality with accurate pipetting at low volumes irrespective of liquid viscosity
  • Enables fast and high-throughput microbiome studies

mosquito from SPT Labtech employ a unique pipetting method, utilizing positive displacement with disposable tips containing a small stainless steel rod in each tip (Figure 1).

This pipetting mechanism enables accurate handling of nanoliter volumes, eliminating the need for specialized liquid classes designated by the instrument control software in contrast to traditional liquid handlers.

mosquito® positive displacement tip technology and mosquito® HV genomics.

Figure 1. mosquito® positive displacement tip technology and mosquito® HV genomics. Image Credit: SPT Labtech

The NEBNext Ultra II FS DNA method was chosen based on its enzymatic fragmentation, which eliminates the need for physical DNA fragmentation at the outset of library preparation.

To assess mosquito HV system's performance, genomic DNA from the ZymoBIOMICS Microbial Community DNA Standard was used as input material, comparing it to manually prepared libraries with varying DNA input amounts.

To determine the system's performance with less standardized input material, reduced-volume NEBNext Ultra II FS DNA libraries were created from individual bacterial isolates and compared to manually prepared Illumina TruSeq Nano libraries prepared from the same input material.

In addition, the NEBNext Ultra II FS DNA automated libraries were compared to TruSeq Nano and TruSeq PCR Free libraries produced either manually or through traditional automation methods.

Manual Vs. Automated

To assess the NEBNext Ultra II FS DNA library kit, the ZymoBIOMICS Microbial Community DNA Standard (Catalog No. D6306) was employed.

This standard comprises a known DNA composition of 10 microbial strains, enabling the evaluation of complete metagenomic workflows. It aids in identifying potential bias and errors that might arise during processing, making it an ideal choice for testing library preparation kits.

It also enabled a direct performance comparison to previously tested library kits utilized at the CGR. Tests were conducted with total input amounts of 50 ng, 1 ng, and 0.1 ng, each in triplicate, as illustrated in Figure 2. 

Schematic representation detailing how NEBNext libraries were generated using the ZymoBIOMICS Microbial Community DNA Standard as input DNA. Manual libraries were made using full volumes according to protocol, automated libraries were made using 1 in 10 volume on the SPT mosquito HV platform.

Figure 2. Schematic representation detailing how NEBNext libraries were generated using the ZymoBIOMICS Microbial Community DNA Standard as input DNA. Manual libraries were made using full volumes according to protocol, automated libraries were made using 1 in 10 volume on the SPT mosquito HV platform. Image Credit: SPT Labtech

Fragment Analyzer traces comparing the size distribution of manual and 1/10 automated libraries for A) 50 ng, B) 1 ng, and C) 0.1 ng of input DNA.

Figure 3. Fragment Analyzer traces comparing the size distribution of manual and 1/10 automated libraries for A) 50 ng, B) 1 ng, and C) 0.1 ng of input DNA. Image Credit: SPT Labtech

Bar chart showing average yields for the manual and 1/10 automated libraries with differing input amounts and PCR cycle numbers. Error bars indicate standard deviation.

Figure 4. Bar chart showing average yields for the manual and 1/10 automated libraries with differing input amounts and PCR cycle numbers. Error bars indicate standard deviation. Image Credit: SPT Labtech

Sequence data were normalized to 13M reads per library, A) indicates average levels of duplicate and mapping percentages, B) shows average fold coverage. Error bars indicate standard deviation.

Figure 5. Sequence data were normalized to 13M reads per library, A) indicates average levels of duplicate and mapping percentages, B) shows average fold coverage. Error bars indicate standard deviation. Image Credit: SPT Labtech

Comparative Performance on Clinical and Bacterial Isolates

While analyses using the ZymoBIOMICS Microbial Community DNA Standard showed promising results, they may not always accurately reflect performance with real-world samples.

Previous data was generated from clinical isolates of bacterial samples using manually prepared TruSeq Nano libraries with 100 ng of input DNA. The same samples were then used for the automated NEBNext Ultra II FS DNA method, employing a 1 in 10 volume approach with 10 ng of input DNA. The results were then compared.

Fragment Analyzer traces comparing the size distribution of manual TruSeq Nano and 1/10 volume automated NEBNext Ultra II FS DNA libraries.

Figure 6. Fragment Analyzer traces comparing the size distribution of manual TruSeq Nano and 1/10 volume automated NEBNext Ultra II FS DNA libraries. Image Credit: SPT Labtech

Bar chart detailing average yields for the manual TruSeq Nano and 1/10 automated NEB libraries generated from clinical bacterial isolates. 100 ng and 10 ng of input DNA were used for the TruSeq Nano and NEB 1/10 libraries, and 8 and 10 cycles of PCR, respectively. Error bars are standard deviation.

Figure 7. Bar chart detailing average yields for the manual TruSeq Nano and 1/10 automated NEB libraries generated from clinical bacterial isolates. 100 ng and 10 ng of input DNA were used for the TruSeq Nano and NEB 1/10 libraries, and 8 and 10 cycles of PCR, respectively. Error bars are standard deviation. Image Credit: SPT Labtech

Sequence data were normalised to 1.3M reads per library. Bar chart shows averages of duplicate and mapping percentages, and fold coverage of the genome. Error bars are standard deviation.

Figure 8. Sequence data were normalized to 1.3M reads per library. Bar chart shows averages of duplicate and mapping percentages and fold coverage of the genome. Error bars are standard deviation. Image Credit: SPT Labtech

Comparison of Automated 1/10 Volume NEB Ultra II FS Libraries to Full Volume Automated and Manual Truseq Nano and Truseq PCR Free Libraries

Previously, workflows in the CGR utilized Illumina TruSeq Nano or TruSeq PCR Free libraries. Data was generated using these methods, both manually and with Beckman full volume automation on the FXP system.

For TruSeq Nano libraries, 100 ng of input DNA was used, while 1 µg of ZymoBIOMICS Microbial Community DNA Standard was employed for TruSeq PCR Free libraries. The data demonstrated comparability among all three methods when 50 ng of DNA was used as input for the 1 in 10 volume NEBNext Ultra II FS DNA libraries.

Sequence data were normalised to 13M reads per library. Bar chart shows averages of A) percentage duplicates, B) percentage mapping, and C) fold coverage (X) of the mock community. Error bars are standard deviation.

Figure 9. Sequence data were normalized to 13M reads per library. Bar chart shows averages of A) percentage duplicates, B) percentage mapping, and C) fold coverage (X) of the mock community. Error bars are standard deviation. Image Credit: SPT Labtech

Conclusion

This article demonstrates that NEBNext Ultra II FS DNA libraries, prepared at a 1 in 10 volume ratio, perform equally well as full-volume manually prepared libraries. Additionally, this approach offers substantial cost savings through the reduction in reaction volumes.

The percentage of duplicates, mapping levels, fold coverage, and GC skew (not displayed here) remained comparable down to 1 ng of input DNA. The entire workflow can be completed within a day, enabling larger projects with more samples and reduced technical bias.

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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Last updated: Oct 23, 2023 at 9:49 AM

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