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Updated: Jan 24, 2026

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing
Published on: March 19, 2018
Triplicate PCR reactions for 16S rRNA gene amplicon sequencing are unnecessary
Clarisse Marotz1, Anukriti Sharma2, Greg Humphrey1
1Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA.
Abstract:
Conventional wisdom holds that PCR amplification for sequencing should employ pooled replicate reactions to reduce bias due to jackpot effects and chimera formation. However, modern amplicon data analysis employs methods that may be less sensitive to such artifacts. Here we directly compare results from single versus triplicate reactions for 16S amplicon sequencing and find no significant impact of adopting a less labor-intensive single-reaction protocol.
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