Metagenomic next-generation sequencing (mNGS) has become a crucial tool in microbiome research, infectious disease studies, and clinical diagnostics. However, optimizing mNGS workflows to get more microbial reads while minimizing costs remains a significant challenge. Common obstacles include high host cell content, sample contamination, inefficient library preparation, and the complexities of bioinformatic analysis. 

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Addressing these challenges can lead to more reliable and actionable results without inflating sequencing costs. Here are four effective ways to achieve more microbial reads for less cost in your mNGS projects:

1. Minimize Host DNA Interference to Get More Microbial Reads

One of the biggest challenges in mNGS is the overwhelming presence of host genetic material, which can dominate sequencing reads and obscure microbial signals. Host interference reduces the sensitivity of detecting pathogens or other microbes and increases the sequencing depth required, driving up costs. Implementing a host depletion step before sequencing can significantly enrich microbial reads as seen in this study. Host depletion allows for a more focused sequencing effort on microbial content, ultimately reducing the number of sequencing reads needed and cutting down on expenses.

The Devin™ Host Depletion filter by Micronbrane Medical offers a novel, efficient approach that overcomes the challenges associated with traditional methods. Read the White Paper!

Nucleated Host Cell Depletion with High Microbial Passing Efficiency

• Utilizes Zwitterionic Interface Ultra-Self-assemble Coating (ZISC) technology to remove 99% of host DNA while allowing over 90% of microbial cells to pass through unaltered. 

Versatility Across Sample Types

• The Devin filter works with a wide range of samples, including complex fluids like blood, without the need for pre-extracted DNA, providing consistent results.

Quick and Simple Workflow

• With our filter host depletion is complete in under 5 minutes, unlike other methods that require lengthy and complex protocols, making Devin ideal for high-throughput and urgent applications.

Cost-Effective and Compatible

• By reducing host cells, deep sequencing is unnecessary, which lowers costs, and enables compatibility with a wide variety of downstream protocols.

2. Streamline mNGS Workflow to Reduce Contamination

Contamination is a persistent problem in mNGS workflows, which can lead to inaccurate results and wasted sequencing runs. Establishing a streamlined, contamination-aware workflow can significantly improve the integrity of sequencing data. This involves using dedicated workspaces, employing stringent contamination controls, and optimizing each step of the workflow to minimize cross-contamination risks. Incorporating system controls and no-template controls (NTCs) is also essential to monitor and detect potential contamination throughout the process, ensuring that any background signals are accounted for. Reducing contamination and using these controls not only increases confidence in your results but also eliminates the need for costly re-sequencing and additional analyses, thereby keeping overall costs low.

Devin Microbial Enrichment Kit 

The Devin Microbial Enrichment Kit addresses contamination issues in mNGS workflows by utilizing ultra-clean, mNGS-grade reagents that significantly reduce the risk of cross-contamination and ensure high-quality sequencing data. By incorporating our mNGS-grade reagents into your workflow, the Devin Kit simplifies the extraction process and enhances the reliability of sequencing results. The Devin Microbial Enrichment Kit can lower the risk of false positives, ultimately reducing costs associated with data validation and quality control, providing you with greater confidence in your findings.

What does mNGS-grade mean? We coined this term because our kits:

This transparency allows you to reduce the risk of unexpected variables impacting your sequencing results.

3. Optimize mNGS Library Preparation for Diverse Sample Types

Library preparation can be challenging especially when dealing with diverse sample types, such as low biomass samples (e.g., respiratory tract or blood) or high microbial count samples (e.g., fecal samples). Low biomass samples often yield insufficient microbial DNA, making it difficult to construct high-quality libraries. On the other hand, fecal samples, while rich in microbial content, can suffer from variability in microbial abundance, requiring careful normalization to ensure consistent and reliable results.

For low biomass samples, techniques that enhance DNA extraction and increase the efficiency of library construction can help capture a more comprehensive microbial profile, reducing the need for costly and repetitive sequencing runs. For higher biomass samples, a kit that incorporates normalization makes balancing microbial abundance easier and prevents dominant species from skewing the results. 

Micronbrane Medical’s Unison Ultralow NGS Library Kit provides a versatile and efficient solution for handling diverse sample types in mNGS workflows. By maximizing sequencing output, improving data reliability, and reducing time and resource consumption. 

Unison uses a proprietary Tn5-based protocol integrating tagmentation and adapter addition into a single step, which reduces handling time and potential errors compared to traditional methods. The result is a consistent library yield and uniform insert sizes (350-550 bp) from a wide range of DNA inputs, ensuring high reproducibility and quality across samples. 

The quality of Unison libraries enhances multiplexing capabilities, allowing more samples to be sequenced in a single run with lower depth. Unison reduces sequencing costs and accelerates turnaround times while maintaining robust pathogen identification with as few as 5 million reads. This efficiency makes it ideal for comprehensive microbiome analysis and targeted applications like antimicrobial resistance profiling.

4. Enhance Bioinformatics Analysis for Accurate Pathogen Detection

Efficient and precise data analysis methods are essential for identifying and quantifying microbial species accurately. Utilizing bioinformatic platforms and algorithms that are specifically tailored for mNGS data can improve the accuracy and sensitivity of pathogen detection. 

Our bioinformatics pipeline was four years in the making and streamlines the analysis of mNGS data using our Pathogen Real-Time Identification by Sequencing (PaRTI-Seq) assay. For researchers, PaRTI-Seq RUO Analysis is available for free with a code in the PaRTI-Seq Kit.

Conclusion

It is possible to get more microbial reads at a lower cost. By minimizing host DNA interference, streamlining workflows to reduce contamination, optimizing library preparation, and enhancing bioinformatic analysis, you can significantly improve the cost-effectiveness and reliability of your mNGS projects.

If you’re looking for an end-to-end solution to address these challenges, consider integrating Micronbrane Medical’s PaRTI-Seq assay into your mNGS workflow. PaRTI-Seq is designed to optimize each of these critical steps, offering an efficient approach to metagenomic sequencing that maximizes microbial reads while minimizing costs.

Contact us to learn how PaRTI-Seq can benefit your mNGS workflow today!