Evaluating the Performance of Oligonucleotide Microarrays for Bacterial Strains with Increasing Genetic Divergence from the Reference Strain

Abstract

DNA oligonucleotide microarrays (oligoarrays) are being developed continuously; however, several issues regarding the applicability of these arrays for whole-genome DNA-DNA strain comparisons (genomotyping) have not been investigated. For example, the extent of false negatives (i.e., no hybridization signal is observed when the amino acid sequence is conserved but the nucleotide sequence has diverged to a level that does not allow hybridization) remains speculative. To provide quantitative answers to such questions, we performed competitive DNA-DNA oligoarray (60-mer) hybridizations with several fully sequenced (tester) strains and a reference strain (whose genome was used to design the oligoarray probes) of the genus Burkholderia and compared the experimental results obtained to the results predicted based on bioinformatic modeling of the probe-target pair using the available sequences. Our comparisons revealed that the fraction of the total probes that provided experimental results consistent with the predicted results decreased substantially with increasing divergence of the tester strain from the reference strain. The fractions were 90.8%, 84.3%, and 77.4% for tester strains showing 96% 89%, and 80% genome-aggregate average nucleotide identity (ANI) to the reference strain, respectively. New approaches to determine gene presence or absence based on the hybridization signal, which outperformed previous approaches (e.g., 92.9% accuracy versus 86.0% accuracy) and to normalize across different array experiments are also described. Collectively, our results suggest that the performance of oligoarrays is acceptable for tester strains showing >90% ANI to the reference strain and provide useful guidelines for using oligoarray applications in environmental gene detection and gene expression studies with strains other than the reference strain.

Design and Verification of a Pangenome Microarray Oligonucleotide Probe Set for Dehalococcoides spp

“…From this, the optimal BSR for accurate prediction of probe cross-hybridization for the strainspecific probes on nontarget genomes was determined to be 83% (see Table S4 in the supplemental material). This finding is in keeping with the recently published determination that a BSR of 83% marks the beginning of meaningful biological hybridization between strains of bacteria (42).…”

“…These parameters of probe performance were examined for intensity values of 1,000 to 1 ϫ 10 6 over intervals of 1,000. From these A second method for determining threshold intensity values was adapted from Oh et al (42). Here, instead of using a single reference genome with designed probes and several tester strain DNA samples, any pair of Dehalococcoides genomes with lower than 90% average nucleotide identity (ANI) was utilized as a reference-tester pair (and vice versa).…”

Genomic Diversity of Escherichia Isolates from Diverse Habitats

“…Validating the performance of microarray experiment is essential for correct interpretation of hybridization data. We thus assessed the performance of oligonucleotide-spotted (spotted) microarrays used in this study based on hybridizations with selected, sequenced tester strains and compared it to the performance of the oligonucleotide, in situ synthesized microarrays used in our previous study, which represent an advanced microarray technology [27] . In brief, we identified the genes shared between the reference and the tester strains based on whole genome sequence comparisons and estimated the expected observed microarray hybridization signal based on the identity between the probe and the target gene in the tester strains using the BLAST score ratio approach, as described previously [27] .…”

Phylogenetic, Microbiological, and Glycoside Hydrolase Diversities within the Extremely Thermophilic, Plant Biomass-Degrading Genus Caldicellulosiruptor

“…In fact, the use of M-CGH can be a useful tool for identifying new isolates of Caldicellulosiruptor species. With additional whole-genome sequences becoming available, a genus-wide oligonucleotide microarray could also be designed to quickly identify the presence of desirable genes encoding GHs and CBMs in novel Caldicellulosiruptor isolates (40). When the growth physiologies of Caldicellulosiruptor species were compared, all species were capable of robust growth on pretreated switchgrass biomass, an important characteristic for CBP.…”