Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates.

Massively parallel pyrosequencing of the small subunit (16S) ribosomal RNA gene has revealed that the extent of rare microbial populations in several environments, the 'rare biosphere', is orders of magnitude higher than previously thought. One important caveat with this method is that sequencing error could artificially inflate diversity estimates. Although the per-base error of 16S rDNA amplicon pyrosequencing has been shown to be as good as or lower than Sanger sequencing, no direct assessments of pyrosequencing errors on diversity estimates have been reported. Using only Escherichia coli MG1655 as a reference template, we find that 16S rDNA diversity is grossly overestimated unless relatively stringent read quality filtering and low clustering thresholds are applied. In particular, the common practice of removing reads with unresolved bases and anomalous read lengths is insufficient to ensure accurate estimates of microbial diversity. Furthermore, common and reproducible homopolymer length errors can result in relatively abundant spurious phylotypes further confounding data interpretation. We suggest that stringent quality-based trimming of 16S pyrotags and clustering thresholds no greater than 97% identity should be used to avoid overestimates of the rare biosphere.

[1]  Harald Meier,et al.  46. ARB: A Software Environment for Sequence Data , 2011 .

[2]  C. Nusbaum,et al.  Quality scores and SNP detection in sequencing-by-synthesis systems. , 2008, Genome research.

[3]  Susan M. Huse,et al.  Accuracy and quality of massively parallel DNA pyrosequencing , 2007, Genome Biology.

[4]  Susan M. Huse,et al.  Microbial diversity in the deep sea and the underexplored “rare biosphere” , 2006, Proceedings of the National Academy of Sciences.

[5]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[6]  Thomas Huber,et al.  Bellerophon: a program to detect chimeric sequences in multiple sequence alignments , 2004, Bioinform..

[7]  K. Schleifer,et al.  ARB: a software environment for sequence data. , 2004, Nucleic acids research.

[8]  Hui-Hsien Chou,et al.  DNA sequence quality trimming and vector removal , 2001, Bioinform..

[9]  S. Dongen Graph clustering by flow simulation , 2000 .

[10]  Norman R. Pace,et al.  Specific Ribosomal DNA Sequences from Diverse Environmental Settings Correlate with Experimental Contaminants , 1998, Applied and Environmental Microbiology.

[11]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[12]  Erko Stackebrandt,et al.  Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology , 1994 .

[13]  E. Stackebrandt,et al.  Nucleic acid techniques in bacterial systematics , 1991 .

[14]  Christus,et al.  A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins , 2022 .