Related Experiment Video
Updated: Jun 4, 2026
08:54
Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications
Published on: November 5, 2020
Reproducibility and quantitation of amplicon sequencing-based detection
Jizhong Zhou1, Liyou Wu, Ye Deng
1Department of Environmental Science and Engineering, Tsinghua University, Beijing, China. jzhou@ou.edu
The ISME Journal
|February 25, 2011
Summary
Amplicon sequencing for microbial communities shows low reproducibility and quantitation due to sampling variability. While useful for detecting community structure differences, caution is advised for quantitative beta-diversity analysis.
Area of Science:
- Microbiology
- Molecular Biology
- Ecology
Background:
- Amplicon sequencing is widely used for microbial community analysis.
- Understanding the reproducibility and quantitation of this method is crucial for reliable ecological studies.
Purpose of the Study:
- To assess the technical reproducibility and quantitation of amplicon sequencing for microbial community structure analysis.
- To evaluate strategies for improving data comparability.
Main Methods:
- Examined 24 microbial communities from a long-term global change experiment.
- Used 16S rRNA gene amplification with barcode tags for technical replicates.
- Included genomic DNA of Shewanella oneidensis MR-1 as a control.
- Investigated strategies like increasing biological replicates and data preprocessing.
Main Results:
- Low technical reproducibility observed, with low operational taxonomic unit (OTU) overlap between replicates.
- High variation in control DNA across samples indicated a lack of quantitation.
- Amplicon sequencing showed substantial effects on beta-diversity estimation but less on alpha-diversity.
- Despite limitations, clear microbial community differences were detected between experimental treatments (warming, clipping).
Conclusions:
- Amplicon sequencing is valuable for analyzing microbial community structure, despite not being fully reproducible or quantitative.
- Careful experimental design and data interpretation are essential, especially for quantitative beta-diversity assessments.
Related Concept Videos
02:57Real Time RT-PCR
Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
The real-time quantification of the number of amplified products is...
03:21RNA-seq
RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...