Haas BJ, Gevers D, Earl AM, et al. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res

Genome Sequencing and Analysis Program, The Broad Institute, Cambridge, Massachusetts 02142, USA.
Genome Research (Impact Factor: 14.63). 02/2011; 21(3):494-504. DOI: 10.1101/gr.112730.110
Source: PubMed


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.

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    • "The representative sequence of each OTU was aligned to the GreenGenes 16S rDNA database using PyNast (Caporaso et al., 2010b). Chimeric sequences were identified by ChimeraSlayer (Haas et al., 2011) and subsequently removed from the OTUs using a python script. A Netwick formatted phylogenetic tree was constructed by employing FastTree (Price et al., 2010). "
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    • "Representative OTUs were aligned using PyNAST (Caporaso et al. 2010) with the default database as a reference. ChimeraSlayer was used to identify and discard chimeric of the successfully aligned reads (Haas et al. 2011). A representative sequence from each OTU was classified directly with the RDP Classifier with a 50 % confidence threshold (Cole et al. 2005). "
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    • "Haas et al. (2011) reported that the number of PCR amplification cycles has a dominant effect on chimera formation. By increasing the PCR extension time, reducing the concentration of template DNA and the number of amplification cycles to the fewest number (approximately 20 cycles) still able to yield sufficient amplicons for sequencing, chimera formation can be alleviated or at least be minimized (Lahr & Katz, 2009; Haas et al., 2011; Stevens et al., 2013). Rapid changes in temperature might produce incomplete products which subsequently anneal to other DNA templates, creating chimeras, thus slowing the PCR ramp speed to 1°C s −1 has been recommended as another modification to inhibit chimera formation (Stevens et al., 2013). "
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