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Microbiome

, 1:10

First Online: 04 March 2013Received: 29 November 2012Accepted: 21 January 2013

Abstract

High-throughput sequencing of 16S rRNA gene amplicons has revolutionized the capacity and depth of microbial community profiling. Several sequencing platforms are available, but most phylogenetic studies are performed on the 454-pyrosequencing platform because its longer reads can give finer phylogenetic resolution. The Pacific Biosciences PacBio sequencing platform is significantly less expensive per run, does not rely on amplification for library generation, and generates reads that are, on average, four times longer than those from 454 C2 chemistry, but the resulting high error rates appear to preclude its use in phylogenetic profiling. Recently, however, the PacBio platform was used to characterize four electrosynthetic microbiomes to the genus-level for less than USD 1,000 through the use of PacBio’s circular consensus sequence technology. Here, we describe in greater detail: 1 the output from successful 16S rRNA gene amplicon profiling with PacBio, 2 how the analysis was contingent upon several alterations to standard bioinformatic quality control workflows, and 3 the advantages and disadvantages of using the PacBio platform for community profiling.

KeywordsPacBio Pacific Biosciences Amplicon 16S rRNA Circular consensus sequence ccs Microbial diversity AbbreviationsASCIIAmerican standard code for information interchange

ccscircular consensus sequences

GCguanine-cytosine

OTUoperational taxonomic unit

PacBioPacific Biosciences

QCquality control

rRNAribosomal RNA

SMRTsingle-molecule real time

USDUnited States dollar

ZMWzero-mode waveguide

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Author: Erin B Fichot - R Sean Norman

Source: https://link.springer.com/article/10.1186/2049-2618-1-10



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