1. Introduction: The Relevance of NGS in Microbiome Research
Next-generation sequencing (NGS) has revolutionized microbiome science, enabling detailed analysis of microbial communities across a wide range of habitats. It offers the depth, resolution, and throughput needed to uncover the structure and function of complex microbial ecosystems – from host-associated niches to environmental samples [1].
As sequencing costs have dropped and bioinformatics tools have advanced, NGS has become central to studies exploring how microbial communities contribute to health, disease, agriculture, and environmental processes. Among these, microbiome research focusing on intestinal microbial populations has emerged as one of the most dynamic and clinically relevant areas.
In this blog post, we offer a practical guide to building a reliable NGS workflow for microbiome research. From sample preparation to sequencing, we address the key steps that influence data quality and reproducibility – and highlight proven solutions that support robust and efficient microbiome analysis.
2. NGS in Microbiome Research: Opportunities and Approaches
NGS has become indispensable in microbiome research due to its ability to profile microbial diversity, detect low-abundance taxa, and reveal functional capabilities through high-throughput sequencing. Two core approaches are commonly used: 16S rRNA gene amplicon sequencing, which targets a conserved bacterial marker gene to generate taxonomic profiles, and shotgun metagenomic sequencing, which sequences all genetic material in a sample to provide both taxonomic and functional insights. Each method offers distinct advantages depending on the required resolution, functional scope, and available resources.
These strategies are applied in diverse microbiome studies, including:
- Gut microbiome research, which explores microbial roles in digestion, immunity, metabolism, and disease.
- Oral microbiome analysis, using saliva or buccal swabs, with implications for oral and systemic health.
- Plant microbiome investigations focused on microbial interactions in roots, leaves, and surrounding soil. (Read more in our blog post: Unlocking the Secrets of Plants and Soil)
- Environmental microbiomes, such as those in soil and aquatic systems, studied for their roles in nutrient cycling, biodiversity, and ecological health.
Integrating Multi-Omics for Deeper Biological Insights
Beyond sequencing, combining NGS data with other molecular layers – such as transcriptomics, proteomics, and metabolomics – provides a more comprehensive understanding of microbial systems. In microbiome research, this often involves pairing metagenomic data with metabolite profiles to explore how microbial gene content shapes metabolic outputs.
A prominent example is the analysis of short-chain fatty acids (SCFAs), microbial metabolites involved in gut barrier integrity, immune modulation, and energy balance. While NGS reveals the composition and metabolic potential of microbial communities, LC-MS and GC-MS are used to quantify SCFAs and other metabolites in targeted and untargeted metabolite analyses.
For further details on combining metagenomics and metabolomics, refer to our blog post: How to Ensure Stool Sample Integrity for Gut Metagenomics & Metabolomics Analysis.
3. How to Set Up an NGS Run?
Setting up a successful next-generation sequencing (NGS) experiment requires careful planning and execution across multiple stages. Each step – beginning with sample collection and ending with data interpretation – plays a critical role in ensuring the quality, reproducibility, and biological relevance of the results. Below, we outline the core components of a standard NGS workflow.
3.1 Sample Collection and Preservation
The foundation of any NGS experiment is high-quality, representative biological material. In microbiome studies, this typically means preserving both microbial composition and nucleic acid integrity from the moment of collection. A major challenge, especially in field studies or remote sampling, is the stabilization of DNA at ambient temperatures.
Invitek Diagnostics provides optimized tools for preserving microbial DNA in diverse sample types:
- Stool samples: The Stool Collection Tube with DNA Stabilizer preserves microbial community composition at room temperature for up to 3 months, maintaining microbial titer and DNA integrity. Additionally, the solution stabilizes key metabolic products such as short-chain fatty acids (SCFAs), making it highly suitable for multi-omics studies.
- Saliva samples: The SalivaGene Collector and SalivaGene Swab Comfort devices are ideal for non-invasive genetic and oral microbiome sampling. They stabilize microbial DNA at room temperature up to 12 months.
Table 2 highlights selected scientific publications demonstrating the use of these products in various microbiome applications.
3.2 Nucleic Acid Extraction
The extraction of high-quality nucleic acids is a pivotal step in any NGS workflow. Especially in microbiome studies, the complexity of the sample matrix – whether stool, saliva, or environmental material – can present significant challenges. Efficient lysis, removal of inhibitors, and consistent yield are essential to ensure downstream library preparation and sequencing success.
Invitek Diagnostics offers a comprehensive portfolio of DNA and RNA extraction kits that are optimized for a wide range of sample types, including stool, saliva, plant material, and environmental matrices such as soil. All kits are developed to deliver high purity and inhibitor-free nucleic acids, fully compatible with the requirements of next-generation sequencing.
For gut and oral microbiome analyses, Invitek Diagnostics provides validated solutions that are widely used in both academic and clinical laboratories. The PSP® Spin Stool DNA Basic Kit and the PSP® SalivaGene DNA Kit are ideal for manual extractions from stool and saliva, respectively, offering robust performance in metagenomic and 16S rRNA gene sequencing workflows. For semi-automated, higher-throughput settings, the InviMag® Stool DNA Kit is recommended. It integrates seamlessly with magnetic particle processors such as the KingFisher™ or the Auto-Pure® product lines and ensures reliable extraction from complex stool samples.
Table 2 highlights selected scientific publications demonstrating the use of these products in various microbiome applications.
3.3 Library Preparation and Clean-Up
Library preparation involves converting extracted DNA into sequencing-ready material through steps like fragmentation, adapter ligation, and amplification. Clean-up is a critical part of this process, removing residual enzymes, primers, and small fragments that can compromise library quality.
The MSB® Spin PCRapace Kit from Invitek Diagnostics offers a fast, two-step clean-up protocol, completing purification in just 7 minutes. Validated for DNA fragments from 80 bp to 30 kb, it ensures high-quality libraries compatible with all major NGS platforms.
Library preparation converts extracted nucleic acids into sequencing-ready libraries through steps such as fragmentation, adapter ligation, and amplification. Each step must be precise to preserve the integrity and representativeness of the input material.
An essential part of this workflow is the clean-up process, which removes enzymes, primers, salts, and unwanted fragments that can interfere with quantification, cluster formation, or sequencing accuracy. Effective clean-up improves both the quality and consistency of the final library.
Invitek Diagnostics offers the MSB® Spin PCRapace Kit as a fast and reliable solution for DNA and PCR product purification. With a simple two-step bind-and-elute protocol, the kit enables complete clean-up in just 7 minutes. It is optimized for DNA fragments in the range of 80 bp – 30 kb and is compatible with all major NGS workflows.
By ensuring clean, high-quality libraries, the MSB® Spin PCRapace Kit supports robust and reproducible sequencing results across a wide range of applications.
These systems offer high accuracy and throughput, though they typically produce shorter reads (50–300 bp), which can be limiting for certain applications such as de novo genome assembly.
3.4 Sequencing Platform Selection
Selecting the right sequencing platform is a key step in planning an NGS experiment. Factors such as read length, throughput, and scalability will influence which platform best suits your microbiome study. While second-generation technologies remain the standard for high-throughput short-read sequencing, third-generation platforms are increasingly used for their long-read capabilities and utility in genome assembly and strain-level resolution.
| TABLE I – Overview of Common NGS Platforms Used in Microbiome Research | |||
|---|---|---|---|
| Platform | Type | Read Length | Key Strengths |
| Illumina | Short‑read (2nd gen) | 75–300 bp | High throughput, high accuracy, broad application scope |
| Ion Torrent | Short‑read (2nd gen) | 200–400 bp | Fast turnaround, cost‑effective for targeted panels |
| MGI | Short‑read (2nd gen) | 100–150 bp | Cost‑efficient alternative, growing global adoption |
| PacBio | Long‑read (3rd gen) | 10–25 kb (HiFi) | Long accurate reads, ideal for genome assembly |
| Oxford Nanopore | Long‑read (3rd gen) | Up to >1 Mb | Real‑time sequencing, portable devices, ultra‑long reads |
All platforms listed above are fully compatible with Invitek Diagnostics’ sample collection devices and extraction kits, providing flexibility across a wide range of workflows. Individual models of each sequencing platform are highlighted in Table 2
3.5 Data Analysis and Interpretation
The final step in an NGS workflow is the analysis and interpretation of sequencing data. In microbiome studies, this involves quality control, removal of low-quality or host-derived reads, and taxonomic or functional profiling using different bioinformatical tools.
4.Demonstrated Compatibility: Insights from the Scientific Literature
Table 2 provides a selection of peer-reviewed publications that demonstrate the successful use of Invitek Diagnostics’ products in microbiome research. These studies cover a variety of sample types and sequencing applications, including human and animal gut, human saliva, plant, and environmental microbiomes.
| TABLE II – Product Compatibility – Sequencing Platforms, Publications & Invitek Products Used | |||
|---|---|---|---|
| Platform |
Publication | Application | Invitek Product(s) used |
| Illumina | |||
| HiSeq1000 | Cockburn AF et al., 2012 | Human Oral Microbiome | SalivaGene Collector; PSP® SalivaGene DNA Kit |
| MiSeq | Hicks et al., 2024 | Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer; PSP® Spin Stool DNA Basic Kit |
| MiSeq | Gemmell et al., 2024 | Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer; PSP® Spin Stool DNA Basic Kit |
| MiSeq | Rötzer et al., 2023 | Animal Gut Microbiome | Stool Collection Tube with DNA Stabilizer |
| MiSeq | Visconti et al., 2024 | Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer; PSP® Spin Stool DNA Basic Kit |
| MiSeq | Merelim et al., 2025 | Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer; PSP® Spin Stool DNA Basic Kit |
| MiSeq | Wylensek et al., 2020 | Animal Gut Microbiome | Stool Collection Tube with DNA Stabilizer |
| MiSeq | Radani et al., 2022 | Human Gut & Oral Microbiome | SalivaGene Collector; Stool Collection Tube with DNA Stabilizer |
| MiSeq | Hauschild et al., 2024 | Bacterial & Fungal Microbiome in Plant & Soil | InviSorb® Spin Plant Mini Kit |
| NextSeq | Bostanci et al., 2021 | Human Oral Microbiome | SalivaGene Collector |
| NextSeq | Krog et al., 2022 | Human Oral Microbiome | SalivaGene Collector |
| NextSeq / NovaSeq | Duru et al., 2025 | Phage & Plasmid Populations in the Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer; PSP® Spin Stool DNA Basic Kit |
| NovaSeq | Liwinski et al., 2024 | Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer |
| Ion Torrent | |||
| Ion Torrent | Sammallahti et al., 2025 | Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer |
| Ion Torrent | Indugu et al., 2017 | Animal Gut Microbiome | PSP® Spin Stool DNA Basic Kit |
| Ion Torrent | Indugu et al., 2016 | Animal Gut Microbiome | PSP® Spin Stool DNA Basic Kit |
| Oxford Nanopore | |||
| MinION | Rathi et al., 2024 | Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer |
| MinION | Pradhan et al., 2023 | Bacterial & Yeast Microbiome | PSP® Spin Stool DNA Basic Kit |
| MinION | Wylensek et al., 2020 | Animal Gut Microbiome | Stool Collection Tube with DNA Stabilizer |
| MGI | |||
| DNBSEQ‑G400/T7 | Fransson et al., 2022 | Human Saliva Microbiome | SalivaGene Collector |
| DNBSEQ‑T7 | Raju et al., 2024 | Human Gut Microbiome | Stool Collection Tube with DNA Stabilizer; PSP® Spin Stool DNA Basic Kit |
| PacBio | |||
| PacBio | Wylensek et al., 2020 | Animal Gut Microbiome | Stool Collection Tube with DNA Stabilizer |
Table 2: Selected publications showing the use of Invitek Diagnostics’ products for sample collection and stabilization and nucleic acid extraction for microbiome applications across all common sequencing platforms. References to each publication are provided at the end of this blogpost.
The publications highlight the reliable performance of Invitek’s solutions for sample stabilization and DNA extraction across a broad range of sequencing platforms, including Illumina, Ion Torrent, MGI, Oxford Nanopore, and PacBio.
5. Summary
Next-generation sequencing has become a cornerstone of microbiome research, enabling in-depth insights into microbial composition and function across a wide range of sample types and environments. From sample collection and stabilization to DNA extraction, library preparation, and sequencing, each step in the workflow plays a vital role in ensuring high-quality, reproducible results.
Invitek Diagnostics offers a comprehensive portfolio of products that support each of these steps. With proven compatibility across all major sequencing platforms, these tools help ensure the reliability and efficiency of NGS workflows in microbiome research.
References
Mardis ER. (2022).DNA sequencing technologies: 2006-2016. Nat Protoc. 2017 Feb;12(2):213-218. doi: 10.1038/nprot.2016.182. Epub 2017 Jan 5. PMID: 28055035.
References of the publications highlighted in Table 2
Bian et al. (2022). Reducing the number of accepted species in Aspergillus series Nigri. Studies in Mycology, 102, 95–132. https://doi.org/10.3114/sim.2022.102.03
Bostanci et al. (2021). Dysbiosis of the Human Oral Microbiome During the Menstrual Cycle and Vulnerability to the External Exposures of Smoking and Dietary Sugar. Frontiers in Cellular and Infection Microbiology. ;11:625229. https://doi.org10.3389/fcimb.2021.625229
Cockburn AF et al. (2012). High throughput DNA sequencing to detect differences in the subgingival plaque microbiome in elderly subjects with and without dementia. Investigative Genetics. 2012 Sep;3(1):19. https://doi.org/10.1186/2041-2223-3-19.
Duru et al. (2025). Comparison of phage and plasmid populations in the gut microbiota between Parkinson's disease patients and controls. Scientific Reports. 2025 Apr;15(1):13723. https://doi.org/10.1038/s41598-025-96924-5.
Fransson et al. (2022). Cohort profile: the Swedish Maternal Microbiome project (SweMaMi) - assessing the dynamic associations between the microbiome and maternal and neonatal adverse events. BMC Pregnancy and Childbirth, 22, 346. https://doi.org/10.1186/s12916-024-03548-z
Gemmell et al. (2024). Optimised human stool sample collection for multi-omic microbiota analysis. Scientific Reports, 14(1). https://doi.org/10.1038/s41598-024-67499-4
Hauschild et al. (2024). Rhizosphere competent inoculants modulate the apple root-associated microbiome and plant phytoalexins. Applied Microbiology and Biotechnology. 2024 May;108(1):344. https://doi.org/10.1007/s00253-024-13181-8
Hicks et al. (2024). Oral, Vaginal, and Stool Microbial Signatures in Patients With Endometriosis as Potential Diagnostic Non-Invasive Biomarkers: A Prospective Cohort Study. BJOG: An International Journal of Obstetrics & Gynaecology. https://doi.org/10.1111/1471-0528.17979
Indugu et al. (2016). A comparison of rumen microbial profiles in dairy cows as retrieved by 454 Roche and Ion Torrent (PGM) sequencing platforms. PeerJ, 4, e1599. https://doi.org/10.7717/peerj.1599
Indugu et al. (2017). Comparison of rumen bacterial communities in dairy herds of different production. BMC Microbiology, 17, 190. https://doi.org/10.1186/s12866-017-1098-z
Krog et al. (2022). The healthy female microbiome across body sites: effect of hormonal contraceptives and the menstrual cycle. Human Reproduction (Oxford, England). 2022 Jun;37(7):1525-1543. https://doi.org/10.1093/humrep/deac094.
Liwinski et al. (2024). Gender-affirming hormonal therapy induces a gender-concordant fecal metagenome transition in transgender individuals. BMC Medicine, 22, 346. https://doi.org/10.1186/s12916-024-03548-z
Merelim et al. (2025). Distinct exercise modalities on GUT microbiome in sarcopenic older adults: study protocol of a pilot randomized controlled trial. Frontiers in Medicine, 12, 1504786. https://doi.org/10.3389/fmed.2025.1504786
Pradhan et al. (2023). Metagenomic and physicochemical analysis of Kombucha beverage produced from tea waste. Journal of Food Science and Technology, 60, 1088–1096. https://doi.org/10.1007/s13197-022-05476-3
Radani et al. (2022) Analysis of Fecal, Salivary, and Tissue Microbiome in Barrett's Esophagus, Dysplasia, and Esophageal Adenocarcinoma. Gastro hep Advances. 2022 ;1(5):755-766. https://doi.org/10.1016/j.gastha.2022.04.003
Raju et al. (2024). Microbial-derived imidazole propionate links the heart failure-associated microbiome alterations to disease severity. Nature Communications, 15, 346. https://doi.org/10.1038/s41467-024-03548-z
Rathi et al. (2024). Study of amino acids absorption and gut microbiome on consumption of pea protein blended with enzymes-probiotics supplement. Frontiers in Nutrition, 11, 1307734. https://doi.org/10.3389/fnut.2024.1307734
Rötzer et al. (2023). Bovine Udder Health: From Standard Diagnostic Methods to New Approaches—A Practical Investigation of Various Udder Health Parameters in Combination with 16S rRNA Sequencing. Microorganisms, 11(5), 1311. https://doi.org/10.3390/microorganisms11051311
Sammallahti et al. (2025). Fecal profiling reveals a common microbial signature for pancreatic cancer in Finnish and Iranian cohorts. Gut Pathogens, 17, Article 24. https://doi.org/10.1186/s13099-025-00698-0
Visconti et al. (2024). Distinct changes in gut microbiota of patients with kidney graft rejection. Nephrology Dialysis Transplantation, 39(Supplement_1), gfae069-0101-1776. https://doi.org/10.1093/ndt/gfae069.101
Wylensek et al. (2020). A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity. Nature Communications, 11, 6389. https://doi.org/10.1038/s41467-020-19929-w
