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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Published on: August 29, 2014
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Primer and platform effects on 16S rRNA tag sequencing.
Julien Tremblay1, Kanwar Singh2, Alison Fern2
1Department of Energy Joint Genome Institute Walnut Creek, CA, USA ; National Research Council Canada Montreal, QC, Canada.
Frontiers in Microbiology
|August 25, 2015
Summary
Comparing 16S rRNA gene sequencing protocols reveals primer choice impacts microbial community abundance. However, sequencing platforms have minimal effects, and beta diversity metrics remain robust across methods.
Area of Science:
- Microbial ecology
- Molecular biology
- Bioinformatics
Background:
- 16S rRNA gene sequencing is widely used for microbial community profiling.
- Variations in protocols (primers, sequencing tech, data processing) exist.
- Comparability of data generated with different protocols is often unclear.
Purpose of the Study:
- To compare the impact of different amplification primers and sequencing technologies on microbial community profiling.
- To assess the comparability of data generated using distinct 16S rRNA gene sequencing protocols.
- To evaluate the influence of protocol choices on quantitative abundance and diversity estimations.
Main Methods:
- Compared three amplification primer sets (V4, V6-V8, V7-V8) and two sequencing technologies (454 pyrosequencing, Illumina MiSeq).
- Utilized a mock community with known species and complex environmental samples.
- Estimated PCR-independent profiles using shotgun sequencing for comparison.
Main Results:
- Illumina MiSeq paired-end reads yielded higher quality data and better read retention than 454 pyrosequencing.
- Primer selection significantly affected quantitative abundance estimations.
- Sequencing platform showed minor effects on results when using matched primers.
- Beta diversity metrics demonstrated robustness against both primer and sequencing platform biases.
Conclusions:
- Protocol choices, particularly primer selection, influence microbial community abundance estimations in 16S rRNA gene sequencing.
- Illumina MiSeq offers advantages in data quality and read retention over 454 pyrosequencing for this application.
- Despite variations, beta diversity analyses are reliable across different 16S rRNA gene sequencing protocols.
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