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Absolute Quantitative 16S/ITS Amplicon Sequencing

Product Introduction

Based on the biases and limitations of conventional amplicon sequencing technology, we have designed 12 specific exogenous internal standard DNAs, which include universal primer binding sites for prokaryotes and eukaryotes, and have been verified by the Blast method to have no match with known natural species sequences. Exogenous internal standard DNAs of known concentration and copy number are added in gradient to the environmental sample DNA, then mixed and amplified, followed by sequencing of the amplicons; during the PCR reaction, the internal standard plasmids will amplify to produce amplicon products of similar size to the 16S-V3V4 or ITS2 fragments. By calculating the absolute abundance of the internal standard DNA and each microbial population in the environmental sample from the sequenced OTUs, a linear regression equation is established between the gradient of internal standard DNA copy numbers (log copies) and the number of sequencing reads (log reads). This equation is used to correct the sample OTU and read data, ultimately yielding information on the composition of the sample community and their absolute abundances.

Technical Parameter

Product	Sequencing Platform	Data Volume
Absolute Quantitative 16S Amplicon Sequencing	NovaSeq PE250	120 K raw reads
Absolute Quantitative ITS Amplicon Sequencing	NovaSeq PE250	120 K raw reads

Product Advantages

More accurate biomass estimation
Enhanced data comparability
Reduced technical bias
Deeper understanding of ecosystem dynamics
Optimized experimental design
Simultaneous absolute + relative quantification analysis

Experimental Process

Joint analysis of absolute quantitative analysis results and relative quantitative analysis results

Analysis Process

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