Cost-effective NGS with automated miniaturization

This article is based on a poster originally authored by Jing Zhang, Martin Däumer, Alexander Thielen, Anna Chanou, and Huw Rees.

Advances in next-generation sequencing (NGS) technology have improved throughput and speed, but the library preparation process continues to present a notable challenge in terms of the manual labor and high cost associated with high-throughput applications.

The mosquito^® liquid handler from SPT Labtech leverages true positive displacement technology to accurately accommodate low volumes (500 nL-5 μL) of samples and reagents, regardless of environmental conditions and liquid viscosity (Figure 1).

This tool has been widely employed in miniaturizing a range of library preparation protocols, with no compromise in accuracy and reducibility.

This article examines the use of mosquito^® HV genomics in automating the Illumina Nextera XT protocol at SEQ-IT. Using this combination, it was possible to achieve 5X miniaturized reaction volumes while continuing to provide reproducible, high-quality libraries.

The development of this miniaturized method has facilitated a range of applications, including identifying and screening mutations with oncogenic potential and drug resistance (HIV, HBV, HCV), SNP detection, and HLA-typing.

Figure 1. mosquito HV genomics liquid handler equipped with 5 deck positions (left), schematic representation of positive replacement technology (right). Image Credit: SPT Labtech

Method

DNA extracted from patient samples was subject to amplicon sequencing, targeting a range from 300 bp to 10 kb across various areas of diagnostic interest. 

SEQ-IT uses the 5X miniaturized Illumina Nextera XT protocol on mosquito^® HV genomics to complete almost 8,000 library preparations per year, equating to around 660 samples per month on average.

Library preparation for 48 samples takes 5.5 hours in total while processing 96 samples requires 6.5 hours, including required PCR cycles. The Illumina MiSeq system is used to perform sequencing.

Figure 2. Graphical representation of the Illumina Nextera XT protocol indicating the pre-PCR steps (pink) and post-PCR steps (teal). Image Credit: SPT Labtech

Table 1. A breakdown of the 50-sample representative sequencing run prepared by the 5X miniaturized Illumina Nextera XT protocol on mosquito HV genomics. Source: SPT Labtech

In the example presented here, a representative sequencing run of 50 samples was prepared and sequenced on MiSeq, using a 500-cycle v2 sequencing reagent in a 2x250 nt mode. Table 1 features a breakdown of the 50 samples. In this instance, the DNA concentration input for tagmentation was 0.2 ng/μL.

Results

Sample library size was analyzed via Bioanalyzer. This was done following amplicon tagmentation and PCR amplification of fragmented DNA (prior to normalization). The results highlighted a typical size distribution with an average size of approximately 500 bp, confirming good tagmentation quality (Figure 3).

Figure 3. The Fragment Analyzer trace shows a typical size distribution of the sample insert pool after tagmentation and bead clean-up. Image Credit: SPT Labtech

It was determined that 99.54% of the reads were correctly mapped (Figure 4), while 84.3% of the sequencing data presented a QC score ≥30. This is in the anticipated range of this specific amplicon approach (Figure 5).

Figure 4. Quality control data shows that 29,200,000 out of 29,333,582 total reads were properly mapped, corresponding to a mapping level of 99.54%. Image Credit: SPT Labtech

Figure 5. 84.3% of the sequencing data presented a QC score ≥30. Image Credit: SPT Labtech

The desired output of each sample had been calculated in advance to evaluate the performance accuracy of mosquito^®. The proportion of the 42 HIV amplicons (2.5 kb) was calculated to match approximately 2% each, while the 6 HIV-1 genomes were expected to have a proportion of 1% each (Figure 6).

It was determined that the proportion of each sample to the total output agreed with the anticipated percentage, confirming the method’s high accuracy.

Figure 6. Proportion of each sample on the total output. The 42 HIV amplicons are presented by blue and the 6 HIV whole genome samples are highlighted by purple. Sample 19 had a low DNA input concentration. The negative controls are indicated by teal asterisks. Image Credit: SPT Labtech

Conclusions

Miniaturizing Illumina Nextera XT library preparation on mosquito has enabled cost savings of up to 80% without compromising sequencing data quality.

A range of metrics was evaluated, including sample fragmentation levels, QC scores, mapping rates, and the distribution of each sample in the total output. All of these metrics demonstrated excellent results, even at 5X miniaturized volume.

It was also highlighted that the entire workflow could be completed within a few hours, enabling higher throughput for larger projects while requiring only minimal sample input.

This miniaturized workflow also decreases the need for manual pipetting, minimizing plastic waste, and ensuring a more cost-effective and environmentally sustainable solution.

Its capacity to address these common bottlenecks means that this approach will enable the more widespread adoption of NGS technologies across a diverse array of applications.

Acknowledgments

Produced from materials originally authored by Jing Zhang, Anna Chanou, and Huw Rees from SPT Labtech; and Martin Däumer and Alexander Thielen from SEQ-IT.

About SPT Labtech

We Design and Manufacture 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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