Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons
Brian J. Haas,Dirk Gevers,Ashlee M. Earl,Mike Feldgarden,Doyle V. Ward,Georgia Giannoukos,Dawn Ciulla,Diana Tabbaa,Sarah K. Highlander,Erica Sodergren,Barbara A. Methé,Todd Z. DeSantis,Joseph F. Petrosino,Rob Knight,Bruce W. Birren +14 more
TLDR
A new chimera detection tool called Chimera Slayer (CS), which detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets.Abstract:
Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.read more
Citations
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UCHIME improves sensitivity and speed of chimera detection
TL;DR: UCHIME has better sensitivity than ChimeraSlayer (previously the most sensitive database method), especially with short, noisy sequences, and in testing on artificial bacterial communities with known composition, UCHIME de novo sensitivity is shown to be comparable to Perseus.
Journal ArticleDOI
UPARSE: highly accurate OTU sequences from microbial amplicon reads
TL;DR: The UPARSE pipeline reports operational taxonomic unit (OTU) sequences with ≤1% incorrect bases in artificial microbial community tests, compared with >3% correct bases commonly reported by other methods.
Journal ArticleDOI
Metagenomic biomarker discovery and explanation
Nicola Segata,Jacques Izard,Jacques Izard,Levi Waldron,Dirk Gevers,Larisa Miropolsky,Wendy S. Garrett,Curtis Huttenhower +7 more
TL;DR: A new method for metagenomic biomarker discovery is described and validates by way of class comparison, tests of biological consistency and effect size estimation to address the challenge of finding organisms, genes, or pathways that consistently explain the differences between two or more microbial communities.
Journal ArticleDOI
Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences
Morgan G. I. Langille,Jesse R. Zaneveld,J. Gregory Caporaso,J. Gregory Caporaso,Daniel McDonald,Dan Knights,Joshua A Reyes,Jose C. Clemente,Deron E. Burkepile,Rebecca Vega Thurber,Rob Knight,Rob Knight,Robert G. Beiko,Curtis Huttenhower,Curtis Huttenhower +14 more
TL;DR: The results demonstrate that phylogeny and function are sufficiently linked that this 'predictive metagenomic' approach should provide useful insights into the thousands of uncultivated microbial communities for which only marker gene surveys are currently available.
Journal ArticleDOI
Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.
TL;DR: This work presents an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads and demonstrates that it can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
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