Analysis of 16S rRNA Amplicon Sequencing Options on the Roche/454 Next-Generation Titanium Sequencing Platform

Hideyuki Tamaki,Jyothi Thimmapuram,Yoichi Kamagata,Chiachi Hwang,Xiangzhen Li,Shiping Wang,Qiaoyan Lin,Chris L Wright,Wen-Tso Liu,Ahmed Moustafa

doi:10.1371/journal.pone.0025263

Abstract

Background16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platformMethodology/Principal FindingsThe following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5–1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management.ConclusionsOur findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

Highlights

Next-generation sequencing technology, in particular pyrosequencing using the Roche/454 platform, has been applied to studies in microbial ecology [1,2,3]
Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output but have little effect on the outcomes of microbial community analysis
This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies

Summary

Introduction

Next-generation sequencing technology, in particular pyrosequencing using the Roche/454 platform, has been applied to studies in microbial ecology [1,2,3]. Pyrosequencing of 16S rRNA genes (16S pyrotagging or 16S pyrosequencing) has virtually replaced the Sanger-based 16S rRNA sequencing method (e.g., clone library) for microbial diversity analysis because it offers several advantages. By using barcoded primers to PCR amplify 16S rRNA genes, microbial communities from multiple samples can be simultaneously examined and compared. A few candidate primers that target the 16S rRNA genes of domain Bacteria have been proposed based on in-silico analysis and/or experimental results [3,4,6,7,8]. 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform

Objectives

Methods

Conclusion