Capturing sequence diversity in metagenomes with comprehensive and scalable probe design.
Ontology highlight
ABSTRACT:
SUBMITTER: Metsky HC
PROVIDER: S-EPMC6587591 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.
2009-10-27 | GSE15832 | GEO
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).
2009-10-26 | E-GEOD-15832 | biostudies-arrayexpress
Project description:Target enrichment sequencing techniques are gaining widespread use in the field of genomics, prized for their economic efficiency and swift processing times. However, their success depends on the performance of probes and the evenness of sequencing depth among each probe. To accurately predict probe coverage depth, a model called Deqformer is proposed in this study. Deqformer utilizes the oligonucleotides sequence of each probe, drawing inspiration from Watson-Crick base pairing and incorporating two BERT encoders to capture the underlying information from the forward and reverse probe strands, respectively. The encoded data are combined with a feed-forward network to make precise predictions of sequencing depth. The performance of Deqformer is evaluated on four different datasets: SNP panel with 38 200 probes, lncRNA panel with 2000 probes, synthetic panel with 5899 probes and HD-Marker panel for Yesso scallop with 11 000 probes. The SNP and synthetic panels achieve impressive factor 3 of accuracy (F3acc) of 96.24% and 99.66% in 5-fold cross-validation. F3acc rates of over 87.33% and 72.56% are obtained when training on the SNP panel and evaluating performance on the lncRNA and HD-Marker datasets, respectively. Our analysis reveals that Deqformer effectively captures hybridization patterns, making it robust for accurate predictions in various scenarios. Deqformer leads to a novel perspective for probe design pipeline, aiming to enhance efficiency and effectiveness in probe design tasks.
| S-EPMC10835675 | biostudies-literature
Project description:Oligonucleotide microarrays have been applied to microbial surveillance and discovery where highly multiplexed assays are required to address a wide range of genetic targets. Although printing density continues to increase, the design of comprehensive microbial probe sets remains a daunting challenge, particularly in virology where rapid sequence evolution and database expansion confound static solutions. Here, we present a strategy for probe design based on protein sequences that is responsive to the unique problems posed in virus detection and discovery. The method uses the Protein Families database (Pfam) and motif finding algorithms to identify oligonucleotide probes in conserved amino acid regions and untranslated sequences. In silico testing using an experimentally derived thermodynamic model indicated near complete coverage of the viral sequence database.
| S-EPMC2248741 | biostudies-literature
Project description:A large proportion of "universal" 16S PCR primers lack sequence homology to many of the "candidate" divisions, severely limiting bacterial diversity assessments. We designed a primer set that offers a 50% increase in silico in coverage of the domain Bacteria over the commonly used primer combination 27F/519R. Comparisons using pyrosequencing on soil environments showed a significant increase in recovery of taxonomic diversity with around a 3-fold increase in recovery of sequences from candidate divisions.
| S-EPMC3406127 | biostudies-literature
Project description:Natural products are universally recognized to contribute valuable chemical diversity to the design of molecular screening libraries. The analysis undertaken in this work, provides a foundation for the generation of fragment screening libraries that capture the diverse range of molecular recognition building blocks embedded within natural products. Physicochemical properties were used to select fragment-sized natural products from a database of known natural products (Dictionary of Natural Products). PCA analysis was used to illustrate the positioning of the fragment subset within the property space of the non-fragment sized natural products in the dataset. Structural diversity was analysed by three distinct methods: atom function analysis, using pharmacophore fingerprints, atom type analysis, using radial fingerprints, and scaffold analysis. Small pharmacophore triplets, representing the range of chemical features present in natural products that are capable of engaging in molecular interactions with small, contiguous areas of protein binding surfaces, were analysed. We demonstrate that fragment-sized natural products capture more than half of the small pharmacophore triplet diversity observed in non fragment-sized natural product datasets. Atom type analysis using radial fingerprints was represented by a self-organizing map. We examined the structural diversity of non-flat fragment-sized natural product scaffolds, rich in sp3 configured centres. From these results we demonstrate that 2-ring fragment-sized natural products effectively balance the opposing characteristics of minimal complexity and broad structural diversity when compared to the larger, more complex fragment-like natural products. These naturally-derived fragments could be used as the starting point for the generation of a highly diverse library with the scope for further medicinal chemistry elaboration due to their minimal structural complexity. This study highlights the possibility to capture a high proportion of the individual molecular interaction motifs embedded within natural products using a fragment screening library spanning 422 structural clusters and comprised of approximately 2800 natural products.
| S-EPMC4406718 | biostudies-literature
Project description:MotivationUniRef databases provide full-scale clustering of UniProtKB sequences and are utilized for a broad range of applications, particularly similarity-based functional annotation. Non-redundancy and intra-cluster homogeneity in UniRef were recently improved by adding a sequence length overlap threshold. Our hypothesis is that these improvements would enhance the speed and sensitivity of similarity searches and improve the consistency of annotation within clusters.ResultsIntra-cluster molecular function consistency was examined by analysis of Gene Ontology terms. Results show that UniRef clusters bring together proteins of identical molecular function in more than 97% of the clusters, implying that clusters are useful for annotation and can also be used to detect annotation inconsistencies. To examine coverage in similarity results, BLASTP searches against UniRef50 followed by expansion of the hit lists with cluster members demonstrated advantages compared with searches against UniProtKB sequences; the searches are concise (∼7 times shorter hit list before expansion), faster (∼6 times) and more sensitive in detection of remote similarities (>96% recall at e-value <0.0001). Our results support the use of UniRef clusters as a comprehensive and scalable alternative to native sequence databases for similarity searches and reinforces its reliability for use in functional annotation.
| S-EPMC4375400 | biostudies-literature
Project description:Phylogenetic diversity--patterns of phylogenetic relatedness among organisms in ecological communities--provides important insights into the mechanisms underlying community assembly. Studies that measure phylogenetic diversity in microbial communities have primarily been limited to a single marker gene approach, using the small subunit of the rRNA gene (SSU-rRNA) to quantify phylogenetic relationships among microbial taxa. In this study, we present an approach for inferring phylogenetic relationships among microorganisms based on the random metagenomic sequencing of DNA fragments. To overcome challenges caused by the fragmentary nature of metagenomic data, we leveraged fully sequenced bacterial genomes as a scaffold to enable inference of phylogenetic relationships among metagenomic sequences from multiple phylogenetic marker gene families. The resulting metagenomic phylogeny can be used to quantify the phylogenetic diversity of microbial communities based on metagenomic data sets. We applied this method to understand patterns of microbial phylogenetic diversity and community assembly along an oceanic depth gradient, and compared our findings to previous studies of this gradient using SSU-rRNA gene and metagenomic analyses. Bacterial phylogenetic diversity was highest at intermediate depths beneath the ocean surface, whereas taxonomic diversity (diversity measured by binning sequences into taxonomically similar groups) showed no relationship with depth. Phylogenetic diversity estimates based on the SSU-rRNA gene and the multi-gene metagenomic phylogeny were broadly concordant, suggesting that our approach will be applicable to other metagenomic data sets for which corresponding SSU-rRNA gene sequences are unavailable. Our approach opens up the possibility of using metagenomic data to study microbial diversity in a phylogenetic context.
| S-EPMC3166145 | biostudies-literature
Project description:We present a web engine boosted fluorescence in-situ hybridization (webFISH) algorithm using a genome-wide sequence similarity search to design target-specific single-copy and repetitive DNA FISH probes. The webFISH algorithm featuring a user-friendly interface (http://www.webfish2.org/) maximizes the coverage of the examined sequences with FISH probes by considering locally repetitive sequences absent from the remainder of the genome. The highly repetitive human immunoglobulin heavy chain sequence was analyzed using webFISH to design three sets of FISH probes. These allowed direct simultaneous detection of class switch recombination in both immunoglobulin-heavy chain alleles in single cells from a population of cultured primary B cells. It directly demonstrated asynchrony of the class switch recombination in the two alleles in structurally preserved nuclei while permitting parallel readout of protein expression by immunofluorescence staining. This novel technique offers the possibility of gaining unprecedented insight into the molecular mechanisms involved in class switch recombination.
| S-EPMC3522715 | biostudies-literature