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MetaTrue-Quant™ Metagenome (Absolute Quantification)

Product Introduction

MetaTrue-Quant™ Metagenome (Absolute Quantification) achieves precise quantification by spiking multiple artificial internal standard fragments with known concentrations and specific synthetic tags into DNA samples (covering three regions, with a total of 79 internal standard fragments). These standards undergo metagenomic library construction and sequencing together with the sample DNA.

Based on the mass ratio of the internal standards added to the sample and the corresponding ratio of sequencing reads obtained for those standards, a key parameter—sequencing yield (Yseq)—is calculated to characterize the overall efficiency of the entire process from DNA input to sequence output.

Using this parameter, sequencing data of target genes can be directly converted into absolute gene copy numbers within the sample, enabling high-throughput, cross-sample comparable absolute quantification without the need for preset primers.

This technology overcomes the limitation of conventional metagenomic sequencing, which provides only relative abundance. By introducing artificial internal standard sequences, it enables accurate calculation of the absolute copy numbers of microbial genes and species in the sample, thereby truly achieving the transition “from proportion to quantity.”

Technical parameters

Product	Sequencing Platform	Data Volume
MetaTrue-Quant™	PE150	10 G Raw Data

Key Advantages

I. Advancement in Internal Standard Design (Multi-concentration, Multi-structure Standards for More Robust Fitting)
(1) Multi-concentration coverage: The internal standards span multiple concentration gradients, accommodating scenarios with large variations in microbial gene abundance across samples. This effectively overcomes the limitations of traditional methods, which have restricted concentration ranges and insufficient resolution at extreme values.
(2) Multi-structure design: The internal standard sequences cover different fragment lengths and GC contents, simulating the structural diversity of microbial DNA in real samples and reducing quantification bias introduced by structural differences.
(3) Method stability validation: Across various complex sample matrices and different sequencing platforms, the unit-mass signal response ratio of the exogenous internal standards remains stable, with systematic variation not exceeding 6.2%. On a logarithmic scale, the correlation coefficient (R²) between measured and theoretically spiked copy numbers of the internal standards is consistently >0.98, demonstrating strong methodological consistency across conditions.

II. Methodological Validation Results (Highly Consistent with qPCR)
(1) Comparable accuracy with no systematic bias: Validation from both absolute quantification and relative abundance perspectives shows no significant difference between MetaTrue-Quant™ and qPCR results (P = 0.56 / 0.59), with dual validation strengthening the reliability of the conclusion.
(2) Higher precision with lower variability: In three replicate samples, the average coefficient of variation (CV) of MetaTrue-Quant™ was 6.7% / 11.8%, significantly lower than that of qPCR (25.8% / 51.8%) (paired t-test, P = 0.03 / 0.01).
(3) High detection concordance with no false positives: The gene qnrS was not detected by either method, demonstrating that MetaTrue-Quant™ possesses specificity comparable to qPCR.

III. System Stability Assessment
(Proprietary model automatically identifies problematic samples—e.g., low DNA purity—to prevent erroneous quantification.)
(1) Independently developed system stability evaluation model: By analyzing the performance of internal standards during the experiment, the model identifies quantification bias caused by sample quality issues (such as low DNA purity) and enables correction of such deviations.
(2) Traditional methods cannot identify these issues and may directly output unreliable results.

IV. Compatibility with Conventional Metagenomic Sequencing
(1) Fully compatible with standard metagenomic sequencing workflows. A single experiment can simultaneously deliver both a relative abundance table and an absolute copy number table—one dataset, two sets of results.

Technical Comparison

Comparison Dimensions	MetaTrue-Quant™（Multi–Internal Standard Consistency-Based Absolute Quantification）	Others (Calibration-Based Absolute Quantification)
Quantification Strategy	Reliable copy numbers are obtained through cross-validation among multiple internal standards under stable sequencing conditions.	Copy numbers are calculated based on a limited number of known concentration calibration points.
Calibration Approach	Multiple internal standards simultaneously participate in calibration and mutually verify each other.	Relies on a small number of calibration points.
Sensitivity to Single-Point Outliers	Cross-validation among multiple internal standards makes the method insensitive to anomalous data points.	A single outlier may significantly affect the results.
Concentration Coverage Range	Covers a wide dynamic range, suitable for samples with coexisting high- and low-abundance targets.	Limited concentration gradient with insufficient capability for extreme-value determination.
Internal Standard Design Concept	In addition to concentration gradients, internal standards encompass different sequence characteristics (length and GC content).	Primarily relies on internal standard concentration as the reference.
Scope of Application	Artificially designed internal standards with broader applicability.	Natural internal standards, potentially constrained by sample background.
Result Reliability Indication	Quantification results are accompanied by reliability and stability indicators.	Only outputs quantification results.
Overall Method Characteristics	Outputs copy numbers while simultaneously evaluating result credibility.	Converts results into copy numbers.

Process Flow

美格基因

Sample Submission Requirements

Sample Type	Sample Submission Amount	Other Requirements
DNA	≥0.5μg	(1)DNA must be free from contamination by RNA, proteins, or other impurities. (2)Filter membrane diameter: 3–4 cm; pore size: 0.22 or 0.45 μm. (3)All nucleic acid samples must be stored in 1.5 mL or 2 mL EP tubes.
Environmental samples such as soil, silt, sediment, etc.	≥3g
Feces	≥2g
Water filter membranes	≥2张

Analysis process

Application Areas

Areas	Typical Samples	Application Value
Environmental Science	Soil, silt, sediment, water filter membranes	Precisely quantify the absolute abundance of functional genes (e.g., nitrogen cycling, carbon degradation) to evaluate the intensity of ecological functions.
Human Health	Feces, intestinal contents	Track changes in the absolute abundance of specific microbial taxa or antimicrobial resistance genes to establish quantitative associations with disease progression.
Agricultural Microbiology	Rhizosphere soil, compost, fertilizers	Monitor the absolute colonization levels of beneficial microorganisms (e.g., nitrogen-fixing bacteria) to guide precision fertilization.
Industrial Fermentation	Fermentation broth, sludge	Real-time monitoring of the absolute copy numbers of key metabolic genes to optimize fermentation processes.
Clinical Samples	Sputum, urine, swabs	Enhance the detection sensitivity of low-abundance pathogens to support the diagnosis of infectious diseases.

Articles Describing the Method and Publications Citing the Method

Article Title	Journal	Year
A Quantitative Metagenomic Sequencing Approach for High Throughput Gene Quantification and Demonstration with Antibiotic Resistance Genes	Applied and Environmental Microbiology	2021
Evaluating Quantitative Metagenomics for Environmental Monitoring of Antibiotic Resistance and Establishing Detection Limits	Environmental Science & Technology	2025
Recommendations for the use of metagenomics for routine monitoring of antibiotic resistance in wastewater and impacted aquatic environments	Critical Reviews in Environmental Science and Technology	2023
A Metagenomic Approach for Characterizing Antibiotic Resistance Genes in Specific Bacterial Populations: Demonstration with Escherichia coli in Cattle Manure	Applied and Environmental Microbiology	2022
A novel synthetic nucleic acid mixture for quantification of microbes by mNGS	Microbial Genomics	2024
A review of the emergence of antibiotic resistance in bioaerosols and its monitoring methods	Reviews In Environmental Science And Bio-technology	2022
Metagenomic Profiling of Pathogens in Simulated Reclaimed Water Distribution Systems Operated at Elevated Temperature Reveals Distinct Response of Mycobacteria to Filtration and Disinfection Conditions	ACS ES&T Water	2023
Methods and challenges for efficient antibiotic discovery from Streptomyces sp.	BIO Web of Conferences	2025
Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp	BMC Genomics	2023

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