Choosing a Metagenomic Sequencing Platform: 7 Key Questions

Choosing the right metagenomic sequencing platform and workflow can be a big decision. It requires you to take a hard look at what you’re hoping to learn, determine what output will provide those insights, and assess the best way to get there.

In a previous blog post, we discussed Tools and Applications for Metagenomic Research. This overview discussed the basic functions of metagenomic sequencing approaches, as well as popular applications and fields of research. In this article, we will break down specifications of metagenomic sequencing technologies with 7 questions you should ask before choosing a platform.

1. What is your research question?

Metagenomics tools have a wide range of target organisms, depth of coverage, and best use cases. When conducting microbial research, you should first identify the research question your project seeks to answer. This will narrow down your technology of choice.

• Metagenomic amplicon sequencing is a species identification tool. This is a targeted taxonomic approach, and can be used to determine what organisms are present in a sample. Think of it as answering: “Who’s here?”

• Metagenomic shotgun sequencing is used to understand the organisms’ functional potential. With a view of metabolic pathways and genes of interest, this technology addresses a “What are they capable of?” research question.

• Metatranscriptome sequencing looks at the RNA present in a sample. This provides insight into the activities of the organisms, answering “What are they doing?”

• Long-read metagenomic sequencing provides complete genome assemblies (MAGs) and full genome structures of organisms. As a relatively new technology, this expands beyond the capabilities of merely identifying microorganisms, moving into a full profile and index of genetic content. Long-read sequencing is also capable of strain phasing, identifying distinctions between extremely similar microbial strains that may be combined into a “consensus” species when using other approaches.

2. What is your organism(s) of interest?

Some metagenomics platforms are better suited for individual or targeted organisms, while others are designed to get a comprehensive look at the community. If you know that you are looking for a specific type of organism, that may also influence which platform is right for you.

• Metagenomic amplicon sequencing targets organisms of interest. Because this approach profiles the 16S, ITS, or 18S genes, it is suitable for research involving bacteria, fungi, or archaea. You can see a list of available standard targeted regions, along with our default primers, here.

• Metagenomic shotgun sequencing looks at all organisms in a sample. This includes bacteria, archaea, fungi, and viruses.

• Metatranscriptome sequencing targets all active organisms. “Active” is the key difference from shotgun sequencing — because this approach focuses on RNA, you will only obtain insights from organisms actively transcribing RNA.

• Long-read metagenomic sequencing can target all organisms. However, this workflow is optimized for bacteria and archaea in Psomagen’s lab.

3. What is your sample type?

Metagenomics projects focus on a wide variety of sample sources. Our labs accept stool, swabs, saliva, urine, and environmental media (including soil and water). More options are available with consultation and preparation for your specific project.

Keep in mind that DNA or RNA extraction from environmental samples, host DNA depletion, and rRNA depletion steps add time to your sequencing project and come at an additional cost. If you intend to submit purified genomic DNA (gDNA), our team can help you determine best practices.

• Metagenomic amplicon sequencing: Bacterial or fungal gDNA.

• Shotgun metagenomic sequencing: DNA or environmental samples with DNA extraction.

• Metatranscriptome: Total RNA.

• Long-read metagenomics: High-molecular-weight (HMW) DNA.

4. How large is your sample?

If you have limited sample volume, this could determine what applications you’re able to run. Sample requirements vary significantly by application. For metagenome amplicon or metagenome shotgun sequencing, we can successfully work with very low-input samples (such as skin swabs). However, for long-read metagenomics (PacBio), high molecular weight (HMW) DNA is essential. Long-read library construction specifically requires at least 500 ng of total DNA.

Large volume samples, often found in biofluid or water sampling projects, may require microbial concentration prior to any sequencing workflow. Our lab team can help you determine if this is necessary for your project.

5. Do you have an instrument preference? 

If you’ve conducted lots of metagenomics sequencing in the past, continuing to run on the same instrument may be important for existing pipelines or datasets.

Even if you’re just getting started, different instruments have their strengths and weaknesses. Short-read platforms, for example, sequence at a much greater depth, while long-read platforms offer significantly longer contigs. Factors such as data comparability, error profiles, assembly goals, cost structure, and downstream expectations often influence platform preference.

In Psomagen’s lab,

• Metagenomic amplicon sequencing is run on the Illumina MiSeq i100 or NextSeq.

• Shotgun metagenomic sequencing and metatranscriptomic projects are run on the Illumina NovaSeq X Plus.

• Long-read metagenomics projects are run on the PacBio Revio.

6. What data output will answer your research question?

Each metagenomic sequencing platform comes with basic data outputs, as well as options for advanced or custom analysis. Selecting your tier of data deliverables is an important step in mapping out your projects’ steps, predicting necessary budget, and ensuring you get the insights you need from your project. Anywhere you choose to run a metagenomics project should clarify deliverables and explain their process for working with you to develop custom analysis.

At Psomagen, all platforms have a standard report available except for metatranscriptome sequencing, which requires custom analysis.

• Metagenomic amplicon sequencing basic data delivery includes FASTQ files, a taxonomy abundance matrix, and diversity (alpha, beta) statistics. For advanced analysis options, we offer Linear discriminant analysis Effect Size (LEfSe) for identifying features (taxa, pathways, or genes) which differentiate biological conditions, as well as Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) used for inferring metabolic function.

• Metagenomic shotgun sequencing basic data delivery includes FASTQ files, a taxonomy abundance matrix, and diversity (alpha, beta) statistics. Reporting includes functional analysis powered by EggNOG, UniRef, MetaCyc, and KEGG databases, as well as antibiotic resistant genes (ARGs) and virulence factors (VFs).

• Metatranscriptome basic data delivery includes FASTQ files, a gene expression table, and a taxonomy abundance matrix. Common choices for metatranscriptome custom reports include differential gene expression (DEGs) and functional analysis powered by KEGG and COG databases. Additional options are available, and can be discussed with our bioinformatics team.

• Long-read metagenomics basic data delivery includes FASTQ (both platforms) and BAM (Revio only) files, high-quality metagenome-assembled genomes (MAGs), and assembly statistics. With additional analysis, reports include MAG annotation, strain phasing, and comparative genomics.

*Additional data output (higher Gb/reads) available upon request

7. Are you limited by budget constraints?

Considering cost per sample, our list of platforms moves from least to most expensive: metagenomic amplicon sequencing < shotgun metagenomics < metatranscriptomics < long-read metagenomics.

If a platform is exactly what you need, but falls outside of your price range, many investigators mitigate budget constraints with adjustments elsewhere — analyzing fewer samples, for example, or conducting some analysis in-house, are ways to reduce the cost of running your project.

Choosing a metagenomics platform is ultimately about alignment — aligning your research question, organism of interest, sample type, data expectations, instrument preference, and budget into a cohesive strategy. No single workflow is “best” in every situation. Instead, each approach offers distinct strengths that serve different scientific goals.

By working through these seven questions before starting your project, you can reduce unnecessary costs, avoid workflow mismatches, and ensure that the data you generate directly answers your scientific question.

Metagenomics is a powerful and rapidly evolving field, and the right platform choice transforms sequencing data from raw reads into meaningful biological insight. If you’re unsure which approach best fits your study design, our team is available to help you move forward with confidence.