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Updated: Mar 14, 2026

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Choice of molecular barcode will affect species prevalence but not bacterial community composition
Karen Lebret1, Joanna Schroeder2, Cecilia Balestreri2
1Marine Biological Association, Citadel Hill, Plymouth, UK; Department of Ecology and Genetics/Limnology, Uppsala University, Uppsala, Sweden.
Abstract:
The rapid advancement of next generation sequencing protocols in recent years has led to the diversification in the methods used to study microbial communities; however, how comparable the data generated from these different methods are, remains unclear. In this study we compared the taxonomic composition and seasonal dynamics of the bacterial community determined by two distinct 16s amplicon sequencing protocols: sequencing of the V6 region of the 16s rRNA gene using 454 pyrosequencing vs the V4 region of the 16s rRNA gene using the Illumina Hiseq 2500 platform. Significant differences between relative abundances at all taxonomic levels were observed; however, their seasonal dynamics between phyla were largely consistent between methods. This study highlights that care must be taken when comparing datasets generated from different methods.
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