Precision Microbiome: An NGS-Based Gut Microbiota Profiling Solution Powered by High-Performance Enzyme Reagents

The Importance of Gut Microbiota Research

The human gut harbors approximately 10¹⁴ microorganisms, whose collective genomic content exceeds the human genome by more than 100-fold. This complex microbial community plays essential roles not only in nutrient metabolism and immune regulation but is also closely linked to a wide range of diseases—including obesity, diabetes, inflammatory bowel disease (IBD), and even depression.

With China’s 14th Five-Year Plan for Bioeconomic Development designating microbiome research as a frontier scientific priority, gut microbiota analysis is rapidly transitioning from basic research toward clinical applications and diagnostics.

 Approaches to Gut Microbiota Research

Level	Key Question	Method	Advantages	Limitations
DNA	Who is there?	Amplicon sequencing / Metataxonome	- Minimal host DNA interference- Well-established reference databases- Low data volume; easy analysis- Low cost, fast	- PCR/primer bias- Only community composition- Usually genus-level resolution
	What can they do?	Metagenomics (mNGS)	- Functional gene profiling- High species-level resolution- Enables MAGs	- Sensitive to host DNA contamination- Large data; complex analysis- High cost, long time
RNA	What are they doing?	Metatranscriptomics	- Captures active genes- Identifies metabolically active taxa- Shows dynamic transcription	- Host RNA contamination- RNA is unstable; difficult- Large data, complex- High cost, long duration
Protein	What are they doing?	Metaproteomics	- Reflects actively translated genes- Reveals protein-level dynamics- Identifies functional microbes	- Host protein interference- High sample complexity- Requires high-end MS- Limited reference databases
Metabolite	What is the functional outcome?	Metabolomics	- Closest to phenotype; good for biomarkers- Studies microbe–host interactions	- Limited coverage per method- Hard to integrate with microbial composition/function

Gut Microbiome NGS Workflow — Challenges & Solutions

Challenges:

• Fecal samples contain PCR inhibitors such as bile salts and humic acids.
• Host-derived human DNA often exceeds 99%, masking microbial signals.
• Library prep kits may leave residual host DNA or background microorganisms from buffers, interfering with detection.
• Gut microbiome has extremely high diversity, including many uncultured species.

To address these specific pain points, Yeasen Biotechnology has developed, through years of optimization, a comprehensive NGS workflow solution specifically designed for gut microbiome analysis.

 

 

Solution 1. Nucleic Acid Extraction

Fecal DNA Extraction

Fecal DNA extraction is a critical step for isolating and purifying microbial genomic DNA from complex stool samples. This step directly impacts the sensitivity and accuracy of subsequent NGS detection due to several challenges: the presence of high concentrations of PCR inhibitors (such as bile salts, humic acids, and polysaccharides), a high background of host-derived human DNA (>99%), and the difficult-to-lyse cell walls of Gram-positive bacteria.

Hieff^TM Stool DNA Kit (Column-Based Method) 18820ES

Key Features

• Wide Applicability: Suitable for feces and intestinal tissues.
• High DNA Yield: Purifies up to 30 µg of genomic DNA from bacteria, viruses, and parasites in a single fecal sample.
• High DNA Purity: The protocol eliminates the need for organic extraction or ethanol precipitation, ensuring the final eluate is free of inhibitors for downstream applications.

Solution 2. 16S rRNA Sequencing

16S rRNA sequencing is a technology that identifies microbial community composition by amplifying and performing high-throughput sequencing on conserved regions (V3-V4 or V4) of the bacterial small subunit ribosomal RNA gene. Independent of culturing methods, this technique can detect unculturable bacteria and serves as the gold standard for analyzing microbial diversity, comparing β-diversity, and discovering biomarkers.

 16S（V3-V4）Bacterial AmpSeq Prep Kit For Illumina 12983ES

Figure 1. Performance of the 16S (V3-V4) Bacterial AmpSeq Prep Kit 12983ES.

(a) Libraries prepared from 1 ng and 10 ng of fecal, soil genomic DNA, and the ZymoBIOMICS Microbial Community DNA Standard showed higher yields with Cat#12983 compared to other kits.

(b) 16S sequencing confirmed accurate detection of microbial gDNA consistent with the expected composition, with Cat#12983 outperforming competitors.

Product Application	Product Name	Cat. No.
Bacterial 16S Profilingfor Illumina	16S (V3-V4) Bacterial AmpSeq Prep Kit ForIllumina	12983ES
	16S (V4) Bacterial AmpSeq Prep Kit For Illumina	12834ES
	Hieff NGSTM Tagment Index Kit for Illumina	17265ES
Bacterial 16S Profiling for MGI	16S (V3-V4) Bacterial AmpSeq Prep Kit For MGI	12833ES
	16S (V4) Bacterial AmpSeq Prep Kit For MGI	12933ES
	Hieff NGS™  Unique Dual Barcode Primer Kit for MGI	13350-13353ES
16S Full-Length Amplification	Hieff NGS™ Multiplex Long PCR Master Mix	17228ES
16S Third-Generation Sequencing	Hieff™ DNA Library Prep Kit for ONT	13301ES

Solution 3. Metagenomic Sequencing

Metagenomics treats the entire microbial community within a specific environment as the research subject and employs high-throughput sequencing technologies to obtain the collective genomic information of the microorganisms.

 Flash OnePot DNA library Preparation kit 12316ES

Figure 2. Detection of DNA of 10 kinds of microorganisms

Libraries were prepared using using the Flash OnePot DNA lib kit （Cat#12316) from 10 ng of the ZymoBIOMICS Microbial Community DNA Standard (Zymo Research #D6306). Libraries were pooled and sequenced on an Illumina MiSeq(2 x 150 bases). and compared across expected and detected composition for both input levels. The detection of specific microbial gDNA was consistent with the expected composition. Expected composition: Cryptococcus neoformans 2%, Saccharomyces cerevisiae 2%, Bacillus subtilis 12%, Escherichia coli 12%, Enterococcus faecalis 12%, Lactobacillus fermentum 12%, Listeria monocytogenes 12%, Pseudomonas aeruginosa 12%, Staphylococcus aureus 12% and Salmonella enterica 12%.

Product Application	Product Name	Cat. No.	Features
Universal Metagenomic Library Prep	Hieff NGS™ OnePot Pro DNA Library Prep Kit V4	12972ES	High resistance to inhibitors with broad sample compatibility Plate-based reagents facilitate high-throughput, automated library preparation
Rapid, Low-DNA Metagenomic Library Prep	Hieff NGS™ OnePot Flash DNA Library Prep Kit	12316ES	Low background noise with ultra-fast 1.75-hour library construction

Solution 4. Metatranscriptomic Sequencing

Metatranscriptomics is a field of study used to investigate the transcriptional activity of diverse microbial organisms within a sample. Unlike traditional genomic sequencing, which focuses on DNA sequences, metatranscriptomic sequencing analyzes the RNA transcripts of microorganisms to reveal their gene expression levels, functional profiles, and metabolic activities.

Product Application	Product Name	Cat. No.	Features
Metatranscriptomic Research	Hieff NGS™ EvoMax RNA Library Prep Kit(dUTP)	12340ES	Plate-based reagents facilitate high-throughput, automated library preparation.
	Hieff NGS™ EvoMax RNA Library Prep Kit(dNTP)	12341ES

Gut microbiome research has entered an era of "multi-technology integration," where NGS provides a blueprint of microbial composition and functional potential. Technological breakthroughs in domestic enzyme raw materials and tool platforms will offer the field more efficient, economical, and controllable solutions, driving precision micro-ecological medicine from "correlative description" to a new stage of "mechanism elucidation" and "clinical intervention."