title: International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.
abstract: This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies.
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, 100 Bureau Dr, Gaithersburg, MD 20899, USA.
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Olson
authors.affiliation: Statistical Engineering Division, National Institute of Standards and Technology, 100 Bureau Dr, Gaithersburg, MD 20899, USA.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Lund
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, 100 Bureau Dr, Gaithersburg, MD 20899, USA.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Zook
authors.affiliation: Instituto de Salud Pública de Chile, Chile.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Rojas-Cornejo
authors.affiliation: American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Beck
authors.affiliation: Science and Innovation Division, LGC, Queens Rd, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Carole
authors.initials: C
authors.lastname: Foy
authors.affiliation: Science and Innovation Division, LGC, Queens Rd, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Jim
authors.initials: J
authors.lastname: Huggett
authors.affiliation: Science and Innovation Division, LGC, Queens Rd, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Whale
authors.affiliation: National Institute of Metrology, Beijing 100013, China.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Sui
authors.affiliation: National Measurement Institute, West Lindfield, NSW 2070, Australia.
authors.forename: Anna
authors.initials: A
authors.lastname: Baoutina
authors.affiliation: National Measurement Institute, West Lindfield, NSW 2070, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Dobeson
authors.affiliation: National Measurement Institute, West Lindfield, NSW 2070, Australia.
authors.forename: Lina
authors.initials: L
authors.lastname: Partis
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, 100 Bureau Dr, Gaithersburg, MD 20899, USA.
authors.forename: Jayne B
authors.initials: JB
authors.lastname: Morrow
keywords: 16S rRNA; DNA sequencing; Interlaboratory study