Revealing microbial species diversity using sequence capture by hybridization.
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ABSTRACT:
SUBMITTER: Marre S
PROVIDER: S-EPMC8767324 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Microorganisms comprise the majority of living organisms on our planet. For many years, exploration of the composition of microbial communities has been performed through the PCR-based study of the small subunit rRNA gene due to its high conservation across the domains of life. The application of this method has resulted in the discovery of many unexpected evolutionary lineages. However, amplicon sequencing is subject to numerous biases, with some taxa being missed, and is limited by the read length of second-generation sequencing platforms, which drastically reduces the phylogenetic resolution. RESULTS:Here, we describe a hybridization capture strategy that allows the enrichment of 16S rRNA genes from metagenomic samples and enables an exhaustive identification and a complete reconstruction of the biomarker. Applying this approach to a microbial mock community and a soil sample, we demonstrated that hybridization capture is able to reveal greater microbial diversity than 16S rDNA amplicon sequencing and shotgun sequencing. The reconstruction of full-length 16S rRNA genes facilitated the improvement of phylogenetic resolution and the discovery of novel prokaryotic taxa. CONCLUSIONS:Our results demonstrate that hybridization capture can lead to major breakthroughs in our understanding of microbial diversity, overcoming the limitations of conventional 16S rRNA gene studies. If applied to a broad range of environmental samples, this innovative approach could reveal the undescribed diversity of the still underexplored microbial communities and could provide a better understanding of ecosystem function.
| S-EPMC5870382 | biostudies-literature
Project description:BackgroundHigh-throughput DNA sequencing technologies have revolutionized genomic analysis, including the de novo assembly of whole genomes from single organisms or metagenomic samples. However, due to the limited capacity of short-read sequence data to assemble complex or low coverage regions, genomes are typically fragmented, leading to draft genomes with numerous underexplored large genomic regions. Revealing these missing sequences is a major goal to resolve concerns in numerous biological studies.MethodsTo overcome these limitations, we developed an innovative target enrichment method for the reconstruction of large unknown genomic regions. Based on a hybridization capture strategy, this approach enables the enrichment of large genomic regions allowing the reconstruction of tens of kilobase pairs flanking a short, targeted DNA sequence.ResultsApplied to a metagenomic soil sample targeting the linA gene, the biomarker of hexachlorocyclohexane (HCH) degradation, our method permitted the enrichment of the gene and its flanking regions leading to the reconstruction of several contigs and complete plasmids exceeding tens of kilobase pairs surrounding linA. Thus, through gene association and genome reconstruction, we identified microbial species involved in HCH degradation which constitute targets to improve biostimulation treatments.ConclusionsThis new hybridization capture strategy makes surveying and deconvoluting complex genomic regions possible through large genomic regions enrichment and allows the efficient exploration of metagenomic diversity. Indeed, this approach enables to assign identity and function to microorganisms in natural environments, one of the ultimate goals of microbial ecology.
| S-EPMC5351058 | biostudies-literature
Project description:Phytoplasmas are insect-vectored, difficult-to-culture bacterial pathogens that infect a wide variety of crop and non-crop plants, and are associated with diseases that can lead to significant yield losses in agricultural production worldwide. Phytoplasmas are currently grouped in the provisional genus 'Candidatus Phytoplasma', which includes 49 'Candidatus' species. Further differentiation of phytoplasmas into ribosomal groups is based on the restriction fragment length polymorphism (RFLP) pattern of the 16S rRNA-encoding operon, with more than 36 ribosomal groups (16Sr) and over 100 subgroups reported. Since disease symptoms on plants are not associated with phytoplasma identity, accurate diagnostics is of critical importance to manage disease associated with these microorganisms. Phytoplasmas are typically detected from plant and insect tissue using PCR-based methods targeting universal taxonomic markers. Although these methods are relatively sensitive, specific and are widely used, they have limitations, since they provide limited resolution of phytoplasma strains, thus necessitating further assessment of biological properties and delaying implementation of mitigation measures. Moreover, the design of PCR primers that can target multiple loci from phytoplasmas that differ at the sequence level can be a significant challenge. To overcome these limitations, a PCR-independent, multilocus sequence typing (MLST) assay to characterize an array of phytoplasmas was developed. Hybridization probe s targeting cpn60, tuf, secA, secY, and nusA genes, as well as 16S and rp operons, were designed and used to enrich DNA extracts from phytoplasma-infected samples for DNA fragments corresponding to these markers prior to Illumina sequencing. This method was tested using different phytoplasmas including 'Ca. P. asteris' (16SrI-B), 'Ca. P. pruni' (16SrIII-A),'Ca. P. prunorum' (16SrX-B), 'Ca. P. pyri' (16SrX-C), 'Ca. P. mali' (16SrX-A), and 'Ca. P. solani' (16SrXII-A). Thousands of reads were obtained for each gene with multiple overlapping fragments, which were assembled to generate full-length (typically >2 kb), high-quality sequences. Phytoplasma groups and subgroups were accurately determined based on 16S ribosomal RNA and cpn60 gene sequences. Hybridization-based MLST facilitates the enrichment of target genes of phytoplasmas and allows the simultaneous determination of sequences corresponding to seven different markers. In this proof-of-concept study, hybridization-based MLST was demonstrated to be an efficient way to generate data regarding 'Ca. Phytoplasma' species/strain differentiation.
| S-EPMC9556853 | biostudies-literature
Project description:The formation water of a deep aquifer (853 m of depth) used for geological storage of natural gas was sampled to assess the mono-aromatic hydrocarbons attenuation potential of the indigenous microbiota. The study of bacterial diversity suggests that Firmicutes and, in particular, sulphate-reducing bacteria (Peptococcaceae) predominate in this microbial community. The capacity of the microbial community to biodegrade toluene and m- and p-xylenes was demonstrated using a culture-based approach after several hundred days of incubation. In order to reveal the potential for biodegradation of these compounds within a shorter time frame, an innovative approach named the solution hybrid selection method, which combines sequence capture by hybridization and next-generation sequencing, was applied to the same original water sample. The bssA and bssA-like genes were investigated as they are considered good biomarkers for the potential of toluene and xylene biodegradation. Unlike a PCR approach which failed to detect these genes directly from formation water, this innovative strategy demonstrated the presence of the bssA and bssA-like genes in this oligotrophic ecosystem, probably harboured by Peptococcaceae. The sequence capture by hybridization shows significant potential to reveal the presence of genes of functional interest which have low-level representation in the biosphere.
| S-EPMC5328808 | biostudies-literature
Project description:Haemophilus influenzae(Hi) is a gram-negative rod shaped bacterium that lives symbiotically in the upper respiratory tract of humans. Capsulated Hi, as well as non-capsulated strains are known to cause a number of significant infections. Recognizing and understanding the links between the phenotypic traits and the genetic background has paramount epidemiological and clinical importance. To date, a panoply of microbiological and molecular biology tools have been developed and utilized by researchers aimed at identifying the evolutionary links among strains and isolates. Comparative genomic hybridization (CGH) has been shown to be a useful tool for screening strains for their genetic content. However, there is a major limitation when CGH is conducted on a microarray based on a single reference genome. CGH results report which genes are present or absent relative to the genome. Hence the information about novel genetic content that the query strain possesses remains obscure. We report here the construction of the first Hi pan-genome microarray representing ca. 4600 features by 70-mers. In addition to those from the Rd strain, new features originate from the unfinished genome sequences present in NCBI database and from our novel gene discovery project efforts using strain HK1212. Genomes of 20strains belonging to different phylogenetic lineages were screened for their gene loss and gain utilizing the species microarray. The results obtained by employing the multistrain species microarray provide comprehensive information about the genomic content of uncharacterized strains. The trees generated by CGH, in general, do not reproduce the phylogeny of a species in terms of vertical evolution, but instead represents the overall relatedness of genomes to one another and provide an assessment about the species genome evolution. Keywords: Comparative Genomic Hybridization
2009-09-09 | GSE8300 | GEO
Project description:Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.
| S-EPMC8110821 | biostudies-literature
Project description:Haemophilus influenzae(Hi) is a gram-negative rod shaped bacterium that lives symbiotically in the upper respiratory tract of humans. Capsulated Hi, as well as non-capsulated strains are known to cause a number of significant infections. Recognizing and understanding the links between the phenotypic traits and the genetic background has paramount epidemiological and clinical importance. To date, a panoply of microbiological and molecular biology tools have been developed and utilized by researchers aimed at identifying the evolutionary links among strains and isolates. Comparative genomic hybridization (CGH) has been shown to be a useful tool for screening strains for their genetic content. However, there is a major limitation when CGH is conducted on a microarray based on a single reference genome. CGH results report which genes are present or absent relative to the genome. Hence the information about novel genetic content that the query strain possesses remains obscure. We report here the construction of the first Hi pan-genome microarray representing ca. 4600 features by 70-mers. In addition to those from the Rd strain, new features originate from the unfinished genome sequences present in NCBI database and from our novel gene discovery project efforts using strain HK1212. Genomes of 20strains belonging to different phylogenetic lineages were screened for their gene loss and gain utilizing the species microarray. The results obtained by employing the multistrain species microarray provide comprehensive information about the genomic content of uncharacterized strains. The trees generated by CGH, in general, do not reproduce the phylogeny of a species in terms of vertical evolution, but instead represents the overall relatedness of genomes to one another and provide an assessment about the species genome evolution. Keywords: Comparative Genomic Hybridization Twenty query strains (denoted HKxxxx) were investigated in this study, with each query strain hybridized against the reference strain, KW20. Two dye-swap experiments were performed, for a total of four hybridizations per query strain. Each 70mer oligo spotted on the H. influenzae species microarray is replicated six times. Positive controls on the array consist of oligos designed from the sequenced reference genome, KW20, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.
2010-10-08 | E-GEOD-8300 | biostudies-arrayexpress
Project description:The biodiversity of fungi, which are extremely important in maintaining the ecosystem balance in alpine lakeside wetlands, has not been fully studied. In this study, we investigated the fungal communities of three lakeside wetlands from different altitudes in the Qinghai-Tibet Plateau and its edge. The results showed that the fungi of the alpine lakeside wetland had higher species diversity. Functional annotation of fungi by FUNGild software showed that saprophytic fungi were the most abundant type in all three wetlands. Further analysis of the microbial phylogenetic molecular ecological network (pMEN) showed that saprophytic fungi are important species in the three wetland fungal networks, while symbiotic fungi and pathotrophic fungi have different roles in the fungal networks in different wetlands. Community diversity was high in all three lakeside wetlands, but there were significant differences in the composition, function and network structure of the fungal communities. Contemporary environmental conditions (soil properties) and historical contingencies (geographic sampling location) jointly determine fungi community diversity in this study. These results expand our knowledge of fungal biodiversity in the alpine lakeside wetlands.
| S-EPMC7284966 | biostudies-literature
Project description:ObjectiveTargeted sequencing of 16S rDNA amplicons is routinely used for microbial community profiling but this method suffers several limitations such as bias affinity of universal primers and short read size. Gene capture by hybridization represents a promising alternative. Here we used a metagenomic extract from the pea aphid Acyrthosiphon pisum to compare the performances of two widely used PCR primer pairs with DNA capture, based on solution hybrid selection.ResultsAll methods produced an exhaustive description of the 8 bacterial taxa known to be present in this sample. In addition, the methods yielded similar quantitative results, with the number of reads strongly correlating with quantitative PCR controls. Both methods can thus be considered as qualitatively and quantitatively robust on such a sample with low microbial complexity.
| S-EPMC6042230 | biostudies-literature
Project description:Cell-free (cf)DNA signatures are quickly becoming the target of choice for non-invasive screening, diagnosis, treatment and monitoring of human tumors. DNA methylation changes occur early in tumorigenesis and are widespread, making cfDNA methylation an attractive cancer biomarker. Already a proven technology for targeted genome sequencing, hybridization probe capture is emerging as a method for high-throughput targeted methylation profiling suitable to liquid biopsy samples. However, to date there are no reports describing the performance of this approach in terms of reproducibility, scalability, and accuracy. In the current study we performed hybridization probe capture using the myBaits® Custom Methyl-seq kit on 172 plasma samples and standards to evaluate its performance on cfDNA methylation analysis. The myBaits® assay showed high target recovery (>90%), demonstrated excellent reproducibility between captures (R 2 = 0.92 on average), and was unaffected by increasing the number of targets in a capture. Finally, myBaits® accurately replicated 'gold standard' beta values from WGBS (average R 2 = 0.79). The results of this study show that custom targeted methylation sequencing with myBaits® offers a cost-effective, reliable platform to profile DNA methylation at a set of discrete custom regions, with potential applicability to liquid biopsies for cancer monitoring.
| S-EPMC9803870 | biostudies-literature