Absolute Quantitative 16s/18s/ITS Amplicon Sequencing

The Introduction of Microorganism Quantification Amplicon Sequencing

16S rRNA gene sequencing is commonly used for identification, classification and quantitation of microbes within complex biological mixture. In brief, the hypervariable regions of 16s rDNA in prokaryote are amplified by PCR. Then the amplicons are sequenced on high-throughput sequencing platform. Data analysis of these unique hypervariable regions is performed to determine the relative abundance of each taxa in a community, and to compare the taxonomic profiling between groups of interest. This level of analysis can help to address changes in the overall microbial profile over time, or between treatment groups. This method thus plays a crucial role in studying the composition and dynamic changes of microbiomes in complex environmental samples.

Recently, the absolute quantification problem of a certain group of microorganisms in a specific environmental sample has attracted more and more attention. In the past, it was detected by absolute quantification of qPCR of specific species, but the results of qPCR are often unstable, and specific primers for specific species qPCR need to be designed and require higher primer specificity. At present, the general 16S rRNA sequencing method can only obtains relative abundance data by the ratio of the number of sequences of a certain OTU to the total number of sequences.

The CD genomics can provide microorganism quantification amplicon sequencing based on 16S external standard sequences to obtain absolute abundance data for species in the sample. The 16S amplicon library is constructed and sequenced by adding a synthetic sequence of known copy number to the sample DNA, and then a standard curve is drawn according to the number of amplicon reads and the absolute copy number of the external standard sequence. And finally the absolute copy number of the 16S rRNA gene of the corresponding species of the OTU representative sequence in the sample is calculated.

Key Features and Advantages

• ≥150,000 reads/sample; Ability to identify novel microbes and novel genes in environmental community samples
• Cost-efficiency
• High accuracy without sample bias
• Reliable and accurate results
• Multiple applications

Project Workflow

	Sample Requirements Sample type: regular soil samples and stool samples, other sample types need to be evaluated Soil ≥ 5 g; Stool ≥ 2 g gDNA amount: > 500ng；gDNA concentration ≥ 20ng/μL
	Sequencing Strategy Recommended region：V3/V4/V3+V4/V4+V5 MiSeq PE250/PE300, HiSeq PE150 or MGI DNBSEQ-T7/DNBSEQ-G400 ≥ 15W reads per sample
	Bioinformatics Analysis Filtering Sequencing data QC Reads assembling, obtain unique tags OTU generation and statistical analysis OTU quantification Species composition analysis Alpha diversity analysis Beta diversity analysis Diversity statistics …… Advanced bioinformatic: Meta analysis Multi-omics integration Clustering algorithms ……

At CD Genomics, with multiple specialists and years of experience in this area, we guarantee you high-quality data and integrated bioinformatics analyses. If you are interested in what CD Genomics can do with the Microorganism Quantification Amplicon Sequencing, please do not hesitate to contact us. We are more than happy to be of assistance!