Schloss PD, Gevers D, Westcott SL.. Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One 6: e27310

Department of Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS ONE (Impact Factor: 3.23). 12/2011; 6(12):e27310. DOI: 10.1371/journal.pone.0027310
Source: PubMed


The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10(6) reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.

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    • "The mothur sequencing analysis platform was used to analyse the resulting data (Schloss et al., 2011). Sequence quality was ensured by trimming reads where the average quality score fell below 35 across a rolling window of 50 bases. "
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    The ISME Journal 11/2015; DOI:10.1038/ismej.2015.198
    • "Those that did not match the primer sequences, were less than 200 bp, or contained any ambiguities were excluded from further analysis. For phylotype analyses, the remaining sequences were denoised (Schloss et al., 2011) and aligned against the SILVA bacterial and archaeal 16S rRNA gene database (Release 115) (Pruesse et al., 2007) in mothur. "
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    ABSTRACT: Intermittent drainage is one of the most promising approaches to mitigate methane (CH4) emission from paddy fields. Irrigated rice fields in Uruguay are temporarily established on soils used as cattle pastures. We studied soil from the pasture-rice rotation (UR) as well as soil from a permanent cattle pasture (UT) hypothesizing that activity and structure of the bacterial and archaeal communities involved in production of CH4 change systematically with intermittent drainage. Methane production started after 7 days and 16 days of anoxic incubation in UR and UT soil, respectively. Then, production rates of CH4 were higher in UT than UR soil. Intermittent drainage significantly decreased the rates of CH4 production. Analysis of δ13C indicated that CH4 was mainly produced from acetate both in UR (73–98%) and UT (51–80%) soil. Intermittent drainage did not change the pathway of CH4 production. Quantitative PCR showed that methanogenic archaeal gene copy numbers (16S rRNA, mcrA) were much lower in UT than UR soil, but increased upon incubation under anoxic conditions. Terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the communities were clearly different between UR and UT soil. The bacterial community consisted of 9 phyla with relative abundance of >1% in both soils. Whereas the archaeal community in UR soil was dominated by Methanocellales and Methanosarcinaceae, that in UT soil was dominated by Crenarchaeota. Anoxic incubation affected the composition of the bacterial and archaeal communities in UT soil, but not so much in UR soil. In UT soil, the relative abundance of Clostridiales increased to 19%, and the archaeal community changed to dominance by Methanosarcinaceae and Methanobacteriales. Subsequent drainage and re-flooding, however, had comparatively little effect on the composition, although it decreased the rates of CH4 production in both soils. Difference in previous soil management and in the structures of the microbial communities apparently only affected their dynamics and functioning after the first flooding but not upon subsequent drainage and re-flooding.
    Soil Biology and Biochemistry 10/2015; 89:238-247. DOI:10.1016/j.soilbio.2015.07.015
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    • "Those that did not match the primer sequences, were less than 200 bp, or contained any ambiguities were excluded from further analysis. For phylotype analyses, the remaining sequences were denoised (Schloss et al., 2011) and aligned against the SILVA bacterial and archaeal 16S rRNA gene database (Release 115) (Pruesse et al., 2007) in mothur. "
    Yang Ji ·

    Soil Biology and Biochemistry 10/2015; 89:238-247.
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