PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample

Abstract

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.Estimation of the extent of PCR-induced artifacts in microbial diversity studies remains an important task in the search for patterns and extent of microbial diversity. The basic types of PCR artifacts have been shown in controlled laboratory studies and can be divided into two categories: those resulting in sequence artifacts (PCR errors), and those skewing the distribution of PCR products due to unequal amplification (PCR bias) or cloning efficiency. Sequence artifacts may arise due to (i) the formation of chimerical molecules (3,10,14,15,25,26,37,38), (ii) the formation of heteroduplex molecules (25,27,29,32), and (iii) Taq DNA polymerase error (4, 25). PCR bias is thought to be due to intrinsic differences in the amplification efficiency of templates (23) or to the inhibition of amplification by the self-annealing of the most abundant templates in the late stages of amplification (31). However, it remains difficult to translate these results to environmental samples in which target genes are orders of magnitude more highly concentrated than in the simple mixtures of templates generally used in controlled laboratory studies.Here, we address the following questions. (i) To what extent do different PCR errors contribute to overestimation of microbial diversity? (ii) Do these PCR errors suggest differences in community structure? (iii) To what extent does PCR bias result in different template distributions after various cycle numbers? Finally, we derive and reiterate recommendations to minimize PCR artifacts.We have recently generated two large 16S rRNA gene libraries (ϳ1,000 sequences each) from a single bacterioplankton sample (1), providing an opportunity to evaluate PCR artifacts in a realistic setting. The first (standard) library was constructed using 35-cycle amplification to mimic commonly used protocols. The second (modified) library was based on the following amplification protocol to reduce the accumulation of PCR artifacts: limitation to 15 cycles of amplification to decrease PCR bias (23) and accumulation of Taq DNA polymerase errors and chimerical sequence formation (25), followed by 3 additional cycles in a fresh reaction mixture (reconditioning PCR step) to minimize the formation of heteroduplex and Taq DNA polymerase errors (32). In addition, we identified Taq DNA polymerase errors in sequences fr...

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