AFLP of the Bacillus cereus group

Amplified Fragment Length Polymorphism (AFLP) (Vos et al. 1995) is a genotyping technique based on the amplification of subsets of genomic restriction fragments using PCR. Genomic DNA is digested with restriction enzymes (usually 2), and double-stranded adapters are ligated to the ends of the DNA fragments to generate template DNA for selective PCR amplification with specific primers. The PCR products are then analyzed by agarose gel electrophoresis to determine the size range of amplified fragments. The presence and absence of fragments defines a fingerprint, or AFLP profile. To increase detection, AFLP has been adapted to use primers labeled with fluorescent dyes, and the resulting fluorescent AFLP profile data consist of the presence or absence of peaks on an electropherogram and the heights of those peaks, where the peak location is analogous to fragment size, and the peak height is analogous to the number of fragments of a given size. Usually, fragments between 100 and 500 bp and the 40 tallest peaks are used for analysis. The genetic relatedness among isolates is determined by comparison of the AFLP profiles (see figure below). In contrast to other widely used used typing methods such as MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Sequence Typing (MLST), which use a small subset of genetic loci, AFLP covers a larger portion of the genome. AFLP has received considerable support for taxonomic studies because it yields a clear delineation of bacteria belonging to the same genomic species, showing a high discriminative power for the differentiation of highly related bacterial strains that belong to the same species. AFLP has been applied to a variety of bacteria, e.g., Pseudomonas syringæ, Mycobacterium, Xanthomonas, and Aeromonas.

A number of studies have employed AFLP, often fluorescent AFLP, for genotypic analysis of strains of B. anthracis and the B. cereus group (Keim et al. 1997, Jackson et al. 1999, Ticknor et al. 2001, Radnedge et al. 2003, Hill et al. 2004, and Guinebretiere et al. 2008). AFLP markers were used to study the molecular evolution and genetic diversity within the monomorphic B. anthracis species, to study the relationships between B. anthracis and closely related B. cereus group strains, and to generate a global phylogenetic analysis of the B. cereus group. In total, 861 B. cereus group isolates have been typed by AFLP. We have recently combined AFLP data with MLST and MLEE typing data using a supertree approach to build a comprehensive phylogenetic view of the B. cereus group population, available in the HyperCAT database containing 3193 B. cereus group isolates.

Below is an overview of the AFLP procedure (for details see Vos et al. 1995 and Ticknor et al. 2001):

AFLP summary picture