A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

Highlights

• SNPs are important diagnostic markers for phylogenetic analysis of Bacillus anthracis.

• The Luminex® xTAG technology offers a high degree of flexibility to test many SNPs.

• Allele-specific dual-priming-oligonucleotides were designed for MOL-PCR assay.

• The 13-plex assay yielded unambiguous SNP calls and lineage affiliation on 73 samples.

• Reproducibility and easy-of-use of the assay were validated by proficiency testing.

Abstract

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2 ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.

Introduction

Bacillus anthracis, the causal agent of anthrax, is an evolutionarily young species that presents very low molecular diversity among strains (Keim et al., 2000, Keim et al., 2009, Pearson et al., 2004). Single nucleotide polymorphisms (SNPs) represent a major source of genetic variation in B. anthracis. Nucleotide substitutions, with mutation rates of approximately 10^− 10 changes per nucleotide per generation, are important biologically informative markers (Vogler et al., 2002). Due to their evolutionary stability, SNPs have been extensively used to determine phylogenetic groups in clonal pathogen species such as B. anthracis and elucidate relationships among worldwide strains (Pearson et al., 2004, Kuroda et al., 2010). By querying a large number of them against collections of diverse strains, a set of 13 representative SNPs that define major clades within the B. anthracis species has been selected and used for assigning an isolate to one sub-lineage or sub-group (Pearson et al., 2004). These canonical SNPs (canSNPs) subdivided all of the B. anthracis isolates into three major lineages (A, B and C), with further subdivisions into 7 distinct sub-lineages (C.Br.A1055, B.Br.KrugerB, B.Br.CNEVA, A.B.r.Ames, A.B.r.Australia94, A.B.r.Vollum, A.B.r.Western North America) and 5 sub-groups (B.Br.001/002, A.B.r.001/002, A.B.r.003/004, A.B.r.005/006, A.B.r.008/009). An additional 14th canSNP that further divides the A.B.r.008/009 group into two sub-groups (A.B.r.008/011 and A.B.r.011/009) was also recently reported (Marston et al., 2011). CanSNP analysis is now considered the reference method in B. anthracis genotyping. Current B. anthracis canSNP typing methods are all based on singleplex real-time technologies, i.e. Dual Probe TaqMan PCR (Van Ert et al., 2007), Mismatch Amplification Mutation Assays (MAMA) (Birdsell et al., 2012) and High Resolution Melting (HRM) (Derzelle et al., 2011).

SNPs are the most common genetic variation found in genomes of all species. It is therefore not surprising that the development of technologies for SNP-based genotyping has been the subject of intense activity (Kwok, 2001, Shi, 2001, Kim and Misra, 2007, Ding and Jin, 2009, Martino et al., 2010). Several platforms and methods now exist (Kwok, 2001, Kim and Misra, 2007), including ultra-high throughput array-based genotyping technologies, such as those offered by Illumina (Gunderson et al., 2006) and Affymetrix (Kennedy et al., 2003), and many other strategies (Germer and Higuchi, 1999, Livak, 1999, Ahmadian et al., 2000, Kutyavin et al., 2000, Mishima et al., 2005, Tobler et al., 2005, Bruse et al., 2008, Edwards et al., 2009). These methods vary largely in their throughput, cost, technical difficulty or laboriousness, subjectivity in allele interpretation and flexibility. But only few of them are at the same time flexible, rapid (< 1 day), cost-effective, and capable of detecting multiplexed signals simultaneously with medium to high throughput (Dunbar, 2006, Bruse et al., 2008, Price et al., 2010). The Luminex bead-based technology is one of these platforms with broad applications on many assay formats, including nucleic acid, receptor–ligand and immune-assays. This suspension array format implies the use of microsphere sets coupled to probes that recognize and bind the target DNAs. Once bound, the target DNA molecules are fluorescently tagged with Streptavidin–R-phycoerythrin, and the beads are individually analyzed by flow cytometry on the Luminex® platform. Each microsphere set is characterized by a distinct spectral address given by the combination of red and infrared fluorophores within the spheres. A red laser recognizes the microsphere set and a green laser provides a quantitative readout of the bound target (Dunbar et al., 2003). Application of Luminex® xTAG technology for multiple SNP genotyping offers several advantages. Based on a suspension of magnetic beads conjugated with probes, the Luminex xTAG microarray format exhibits rapid hybridization kinetics, flexibility in assay design and is cost-effective (Dunbar, 2006). It can also be compiled as desired by adding or replacing beads and probes without having to reformat and print new arrays (Janse et al., 2012). This system has been increasingly used for the design of several diagnostic assays based on various approaches, namely, Direct Hybridization, Allele-Specific Primer Extension, Single-Base-Extension and Oligonucleotide Ligation (Lee et al., 2004, Dunbar, 2006, Ducey et al., 2007, Ward et al., 2008, Stucki et al., 2012).

In 2010, a smart and more flexible assay method than any other approaches adapted to the Luminex® platform has been introduced by Deshpande et al. for successful pathogen detection and SNP-typing of B. anthracis, Yersinia pestis, and Francisella tularensis (Deshpande et al., 2010). Conceptually related to Multiplex Ligation-dependent Probe Amplification (MLPA) technique (Schouten et al., 2002), this method, called Multiplex Oligonucleotide Ligation-PCR (MOL-PCR), enables direct detection of multiple nucleic acid signatures in a single tube reaction (Deshpande et al., 2010). In MOL-PCR, detection probes consist of modular components that enable target detection, probe amplification, and subsequent capture onto microsphere arrays. MOL-PCR uses allele-specific ligation for allele discrimination, singleplex PCR for signal amplification and hybridization to fluorescent microspheres (beads) for signal detection on a flow cytometer. The ability to discriminate base pair mismatches flanking the ligation site is conferred by the ligase used in the reaction. An enhanced MOL-PCR procedure that makes the method easier to perform than similar published methods to carry out SNP multilocus genotyping has recently been described (Stucki et al., 2012).

In the present work, we adapted and modified the MOL-PCR method for the simultaneous typing of a set of 13 B. anthracis lineage-specific canSNPs previously identified. The developed multiplex assay was primarily built on the MOL-PCR concept as described by Stucki et al. (2012) but with a key modification for SNP discrimination and oligonucleotide design improvement. A dual priming oligonucleotide (DPO) system (Chun et al., 2007) was coupled to the MOL-PCR method to increase assay specificity and allow multiplexing reactions to be more easily designed and produced. The setup and validation of a 13-plex SNP-typing assay for the identification of the main phylogenetic lineages of B. anthracis are presented.