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DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes.

ABSTRACT: DNA microarrays constructed with full length ORFs from Shewanella oneidensis, MR-1, were hybridized with genomic DNA from nine other Shewanella species and Escherichia coli K-12. This approach enabled visualization of relationships between organisms by comparing individual ORF hybridizations to 164 genes and is further amenable to high-density high-throughput analyses of complete microbial genomes. Conserved genes (arcA and ATP synthase) were identified among all species investigated. The mtr operon, which is involved in iron reduction, was poorly conserved among other known metal-reducing Shewanella species. Results were most informative for closely related organisms with small subunit rRNA sequence similarities greater than 93% and gyrB sequence similarities greater than 80%. At this level of relatedness, the similarity between hybridization profiles was strongly correlated with sequence divergence in the gyrB gene. Results revealed that two strains of S. oneidensis (MR-1 and DLM7) were nearly identical, with only 3% of the ORFs hybridizing poorly, in contrast to hybridizations with Shewanella putrefaciens, formerly considered to be the same species as MR-1, in which 63% of the ORFs hybridized poorly (log ratios below -0.75). Genomic hybridizations showed that genes in operons had consistent levels of hybridization across an operon in comparison to a randomly sampled data set, suggesting that similar applications will be informative for identification of horizontally acquired genes. The full value of microbial genomic hybridizations lies in providing the ability to understand and display specific differences between closely related organisms providing a window into understanding microheterogeneity, bacterial speciation, and taxonomic relationships.

SUBMITTER: Murray AE

PROVIDER: S-EPMC55542 | biostudies-literature | 2001 Aug

REPOSITORIES: biostudies-literature

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DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes.

Murray A E AE Lies D D Li G G Nealson K K Zhou J J Tiedje J M JM

Proceedings of the National Academy of Sciences of the United States of America 20010807 17

DNA microarrays constructed with full length ORFs from Shewanella oneidensis, MR-1, were hybridized with genomic DNA from nine other Shewanella species and Escherichia coli K-12. This approach enabled visualization of relationships between organisms by comparing individual ORF hybridizations to 164 genes and is further amenable to high-density high-throughput analyses of complete microbial genomes. Conserved genes (arcA and ATP synthase) were identified among all species investigated. The mtr op ...[more]

PMID: 11493693

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Project description:BackgroundIterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific iodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA) genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes.ResultsIn the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands > or = 12b that are conserved between primates and fish.ConclusionExperimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a re-sequencing microarray when their sequences differ by more than a few percent. The data suggest that interspecific cross-hybridization will not interfere with the accurate recovery of species-specific data from multispecies microarrays, provided that the species' DNA sequences differ by > 20% (mean of 5b differences per 25b oligo). Recovery of DNA sequence data from multiple, distantly-related species on a single multiplex gene chip should be a practical, highly-parallel method for investigating genomic biodiversity.

Project description:The wide applications of next-generation sequencing (NGS) technologies in metagenomics have raised many computational challenges. One of the essential problems in metagenomics is to estimate the taxonomic composition of a microbial community, which can be approached by mapping shotgun reads acquired from the community to previously characterized microbial genomes followed by quantity profiling of these species based on the number of mapped reads. This procedure, however, is not as trivial as it appears at first glance. A shotgun metagenomic dataset often contains DNA sequences from many closely-related microbial species (e.g., within the same genus) or strains (e.g., within the same species), thus it is often difficult to determine which species/strain a specific read is sampled from when it can be mapped to a common region shared by multiple genomes at high similarity. Furthermore, high genomic variations are observed among individual genomes within the same species, which are difficult to be differentiated from the inter-species variations during reads mapping. To address these issues, a commonly used approach is to quantify taxonomic distribution only at the genus level, based on the reads mapped to all species belonging to the same genus; alternatively, reads are mapped to a set of representative genomes, each selected to represent a different genus. Here, we introduce a novel approach to the quantity estimation of closely-related species within the same genus by mapping the reads to their genomes represented by a de Bruijn graph, in which the common genomic regions among them are collapsed. Using simulated and real metagenomic datasets, we show the de Bruijn graph approach has several advantages over existing methods, including (1) it avoids redundant mapping of shotgun reads to multiple copies of the common regions in different genomes, and (2) it leads to more accurate quantification for the closely-related species (and even for strains within the same species).

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DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes.

Publications

DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes.

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