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.

Genomic Prediction of Resistance to Pasteurellosis in Gilthead Sea Bream (Sparus aurata) Using 2b-RAD Sequencing

“…Quality and adapters trimming of sequenced reads were performed by running a customized script (Pauletto et al 2016; Pecoraro et al 2016), obtaining 34-bp long fragments. SNP calling was performed using STACKS v1.23 (Catchen et al 2013).…”

Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

“…This RAD sequencing technique has been used to identify candidate genes associated with specific traits (Luo et al, 2017), to construct ultra‐high density genetic maps (Fu, Liu, Yu, & Tong, 2016), to identify genomic regions under selection in population genetic studies (Pecoraro et al, 2016), and to perform genomic prediction for relevant traits in agricultural species (Palaiokostas, Ferraresso, Franch, Houston, & Bargelloni, 2016). It has also been extended to microbial ecology (Pauletto et al, 2016).…”

High-resolution linkage and quantitative trait locus mapping using an interspecific cross between Argopecten irradians irradians (♀) and A. purpuratus (♂)

“…Of these techniques, an optimized representative restriction site-associated DNA (RAD) method, 2b-RAD, has gained popularity owing to its simplicity in library preparation and ability to provide even and tunable genome coverage with a low requirement of sample quality (Wang et al 2012. This method has been successfully applied for genetic and breeding studies, such as highdensity linkage map construction and QTL mapping, in a wide range of species (Barfield et al 2016;Cui et al 2015;Dixon et al 2015;Guo et al 2014;Jiao et al 2013;Lowry et al 2015;Pauletto et al 2016;Seetharam and Stuart 2013;Tian et al 2015).…”