title: Extending RAD tag analysis to microbial ecology: a comparison between MultiLocus Sequence Typing and 2b-RAD to investigate Listeria monocytogenes genetic structure.
abstract: The advent of next-generation sequencing (NGS) has dramatically changed bacterial typing technologies, increasing our ability to differentiate bacterial isolates. Despite it is now possible to sequence a bacterial genome in a few days and at reasonable costs, most genetic analyses do not require whole-genome sequencing, which also remains impractical for large population samples due to the cost of individual library preparation and bioinformatics. More traditional sequencing approaches, however, such as MultiLocus Sequence Typing (mlst) are quite laborious and time-consuming, especially for large-scale analyses. In this study, a genotyping approach based on restriction site-associated (RAD) tag sequencing, 2b-RAD, was applied to characterize Listeria monocytogenes strains. To verify the feasibility of the method, an in silico analysis was performed on 30 available complete genomes. For the same set of strains, in silico mlst analysis was conducted as well. Subsequently, 2b-RAD and mlst analyses were experimentally carried out on 58 isolates collected from food samples or food-processing sites. The obtained results demonstrate that 2b-RAD predicts mlst types and often provides more detailed information on population structure than mlst. Moreover, the majority of variants differentiating identical sequence type isolates mapped against accessory fragments, thus providing additional information to characterize strains. Although mlst still represents a reliable typing method, large-scale studies on molecular epidemiology and public health, as well as bacterial phylogenetics, population genetics and biosafety could benefit of a low cost and fast turnaround time approach such as the 2b-RAD analysis proposed here.
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro, Italy.
authors.forename: Marianna
authors.initials: M
authors.lastname: Pauletto
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro, Italy.
authors.forename: Lisa
authors.initials: L
authors.lastname: Carraro
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro, Italy.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Babbucci
authors.affiliation: Istituto Zooprofilattico delle Venezie, Viale dell'Università 10, 35020, Legnaro, Italy.
authors.forename: Rosaria
authors.initials: R
authors.lastname: Lucchini
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Bargelloni
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Cardazzo
mesh_terms: D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004783:Environmental Microbiology; D005516:Food Microbiology; D014644:Genetic Variation; D005828:Genetics, Population; D008089:Listeria monocytogenes / Q000145:classification* / Q000235:genetics*; D058889:Molecular Typing / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes
keywords: 2b-RAD; Listeria monocytogenes; bacterial typing; next-generation sequencing; population structure