Microbial metagenomics—assaying the identity and quantity of microbial sequences in a sample—is a powerful sequencing technique that provides valuable insight into the microbial diversity present within environmental samples such as soil or water, as well as within stool and tissue of living organisms. Shotgun metagenomics, an increasingly popular method in this relatively new field, involves sequencing the total DNA present in a sample in an unbiased and sample-agnostic manner. This approach has advantages over older methods such as 16s rDNA or ITS sequencing that have been used historically for microbial strain identification using more targeted approaches. The systematic use of shotgun metagenomics has transformed our understanding of the world around us and scientists are just starting to unlock its potential.1
Like many NGS library prep applications, preparing metagenomic samples for high-throughput DNA sequencing can be a costly, labor-intensive and time-consuming procedure. With a high cost per sample, researchers are often limited when undertaking larger-scale project goals. And as the need for metagenomic sequencings continues to grow, scientists must contend with balancing the necessity for high-quality data and the cost of obtaining such information.
To address the challenge of creating scalable methods for shotgun metagenomics, new library prep methods often focus on ways to reduce costs or workflow burdens. Different types of approaches tackle this challenge in different ways:
- Miniaturization or dilution methods modify existing NGS library kit protocols and methods to allow more samples to be processed per kit unit
- Newer technologies, such as seqWell’s plexWell™ 96, provide assay-ready 96-well (or higher) configurations to streamline high-throughput multiplexed library preparation
The accuracy of these new methods as compared to using standard NGS library products is an important question that can help researchers understand any potential performance or workflow tradeoffs associated with reducing material cost.1
Comparing Miniaturized Library Prep to Standard Approaches
In a recent study by Gaulke, et al., published in the American Society of Microbiology Journal, researchers assessed the performance of miniaturized library prep procedures compared to standard approaches. In this study, five shotgun DNA library prep methods from a range of commercially available kits were evaluated, and each method’s ability to produce metagenomic data accurately was measured across a range of microbial community types, varying in complexity and quantity of input DNA.
The products used in this study compared Illumina’s Nextera DNA Flex, a modified DNA Flex protocol (Illumina), Qiagen’s QIASeq FX DNA, PerkinElmer’s NextFlex Rapid DNA-Seq 2.0, and seqWell’s plexWell 96 library preparation method. The samples that were analyzed represented DNA isolates from a range of microbial community types and mock specimens: low-complexity communities (ocean coral microbiomes), moderately complex communities (mouse stool), and a highly complex community (soil).
The study authors found that the products and methods tested had different levels of precision (accuracy) and ability to recapitulate the known mock samples, but interestingly, the only two methods that performed “good” or higher across all sample types were the Nextera Flex (reduced prep) and plexWell 96 methods.
Methods were also evaluated based on cost. The authors found that the most economical choices for metagenomic library generation were Nextera Flex (reduced prep) and plexWell 96. 1 In contrast, the standard non-reduced Nextera DNA Flex full preparation was more costly than the other methods tested, with NextFlex Rapid XP and QIASeq FX falling in the medium price range.
The study authors also rated the different methods by other factors, such as workflow speed, automation-friendliness, and here, other differences between the kits emerge. For example, while the Nextera Flex (reduced prep) method produces consistently high score for precision and accuracy on the mock samples, it is also more time consuming and less automation-friendly.
Of all of the methods of the study, only the plexWell 96 kit was rated in the top category on speed (hands-on time), automation-friendliness and cost while still being rated as “good” or higher in terms of precision and accuracy on the real and mock community samples.
The American Society of Microbiology Journal study’s findings underscore the importance of carefully choosing a metagenomic library preparation method that takes into account sample properties including community type, composition, and DNA amount.1
So, what does this all mean for your research? First, it is apparent that the methods evaluated in this study produced a range of taxonomic and gene family diversity profiles. Users should be aware that not every kit is equally capable of producing high-quality data on every sample type. Beyond that, even for kits that are capable of producing best-in-class data, there can be significant differences in workflow speed or automation-friendliness. Reduced-format modification or miniaturization, while an attractive cost-saving approach, can have trade-offs in other areas, such as set up time or simplicity of automation, and these are important factors to consider when assessing different approaches.
The plexWell 96 kit is a cost-effective and scalable alternative, and is easier and faster for most users who are considering using a reduced or miniaturized format of an off-the-shelf kit. With plexWell library prep technology, well-balanced libraries for up to 96 samples can be efficiently multiplexed in a single tube, and 100s of incoming samples can be readied for sequencing with minimal hands-on time. This could lead to big improvements in throughput when it comes to metagenomic investigations. 1