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International Interlaboratory Study Comparing Single Organism 16S rRNA Sequencing Data: Going Beyond Consensus Sequence Comparisons



Nathanael D. Olson, Justin M. Zook, Steven P. Lund, Fabiola Rojas-Cornejo, Jim Huggett, Jayne B. Morrow


The Consultative Committee for Amount of Substance established a working group to advance the metrology of microbial identification and quantification. This study represents the initial step towards international comparability and measurement assurance for microbial identity. Six laboratories sequenced the 16S rRNA gene from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes strain 4b NCTC 19944. Participants used either Sanger sequencing, Roche 454 pyrosequencing® (“454”), or Ion Torrent PGM®. The 16S rRNA gene is a multi-copy gene with both conserved and variable bases. To rigorously challenge comparability, the sequencing data was evaluated on three levels: 1) identity of conserved bases, 2) ratio of bases at the variable positions, and 3) the full-length sequences of the individual gene copies. Regardless of sequencing method the biologically conserved positions were correctly identified. To determine the base copy ratio and the full-length gene copy sequences, analytical methods were developed using Bayesian and Maximum Likelihood statistics. Accurate base copy ratios were only determined using datasets with higher coverage, or a greater number of sequencing reads covering the reference gene sequence. However, the base copy ratio was predicted accurately more frequently for lower coverage Sanger clone libraries compared to higher coverage Ion Torrent data. The full-length gene copy sequences were only accurately predicted with “454” datasets, indicating that longer reads and higher throughput are required to determine the full-length gene copy sequences. This study presents novel methods for comparing sequencing data from different sequencing platforms for a multi-copy gene, paving the way towards measurement assurance for DNA sequence based microbial identification.
PLoS One


16S rRNA, microbial identification, DNA sequencing, interlaboratory study


Olson, N. , Zook, J. , Lund, S. , Rojas-Cornejo, F. , Huggett, J. and Morrow, J. (2015), International Interlaboratory Study Comparing Single Organism 16S rRNA Sequencing Data: Going Beyond Consensus Sequence Comparisons, PLoS One, [online], (Accessed July 17, 2024)


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Created March 5, 2015, Updated November 10, 2018