Short DNA Sequences Research Articles

Integrating Genetic and Chromosome Maps of Allium cepa: From Markers Visualization to Genome Assembly Verification.

The ability to directly look into genome sequences has opened great opportunities in plant breeding. Yet, the assembly of full-length chromosomes remains one of the most difficult problems in modern genomics. Genetic maps are commonly used in de novo genome assembly and are constructed on the basis of a statistical analysis of the number of recombinations. This may affect the accuracy of the ordering and orientation of scaffolds within the chromosome, especially in the region of recombination suppression. Moreover, it is impossible to assign contigs lacking DNA markers. Here, we report the use of Tyr-FISH to determine the position of the short DNA sequence of markers and non-mapped unique copy sequence on the physical chromosomes of a large-genome onion (Allium cepa L.). In order to minimize potential background masking of the target signal, we improved our earlier developed pipeline for probe design. A total of 23 markers were located on physical chromosomes 2 and 6. The order of markers was corrected by the integration of genetic, pseudochromosome maps and cytogenetic maps. Additionally, the position of the mlh1 gene, which was not on the genetic map, was defined on physical chromosome 2. Tyr-FISH mapping showed that the order of 23.1% (chromosome 2) and 27.3% (chromosome 6) of the tested genes differed between physical chromosomes and pseudochromosomes. The results can be used for the improvement of pseudochromosome 2 and 6 assembly. The present study aims to demonstrate the value of the in situ visualization of DNA sequences in chromosome-scaffold genome assembly.

Genome Protection by DNA Polymerase θ.

DNA polymerase θ (Pol θ) is a DNA repair enzyme widely conserved in animals and plants. Pol θ uses short DNA sequence homologies to initiate repair of double-strand breaks by theta-mediated end joining. The DNA polymerase domain of Pol θ is at the C terminus and is connected to an N-terminal DNA helicase-like domain by a central linker. Pol θ is crucial for maintenance of damaged genomes during development, protects DNA against extensive deletions, and limits loss of heterozygosity. The cost of using Pol θ for genome protection is that a few nucleotides are usually deleted or added at the repair site. Inactivation of Pol θ often enhances the sensitivity of cells to DNA strand-breaking chemicals and radiation. Since some homologous recombination-defective cancers depend on Pol θ for growth, inhibitors of Pol θ may be useful in treating such tumors.

Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa.

Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa.

PROBER identifies proteins associated with programmable sequence-specific DNA in living cells.

DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

Hidden Addressing Encoding for DNA Storage.

DNA is a natural storage medium with the advantages of high storage density and long service life compared with traditional media. DNA storage can meet the current storage requirements for massive data. Owing to the limitations of the DNA storage technology, the data need to be converted into short DNA sequences for storage. However, in the process, a large amount of physical redundancy will be generated to index short DNA sequences. To reduce redundancy, this study proposes a DNA storage encoding scheme with hidden addressing. Using the improved fountain encoding scheme, the index replaces part of the data to realize hidden addresses, and then, a 10.1 MB file is encoded with the hidden addressing. First, the Dottup dot plot generator and the Jaccard similarity coefficient analyze the overall self-similarity of the encoding sequence index, and then the sequence fragments of GC content are used to verify the performance of this scheme. The final results show that the encoding scheme indexes with overall lower self-similarity, and the local thermodynamic properties of the sequence are better. The hidden addressing encoding scheme proposed can not only improve the utilization of bases but also ensure the correct rate of DNA storage during the sequencing and decoding processes.

TDTHub, a web server tool for the analysis of transcription factor binding sites in plants.

SUMMARYTranscriptional regulation underlies most developmental programs and physiological responses to environmental changes in plants. Transcription factors (TFs) play a key role in the regulation of gene expression by binding specifically to short DNA sequences in the regulatory regions of genes: the TF binding sites (TFBSs). In recent years, several bioinformatic tools have been developed to detect TFBSs in candidate genes, either by de novo prediction or by directly mapping experimentally known TFBSs. However, most of these tools contain information for only a few species or require multi‐step procedures, and are not always intuitive for non‐experienced researchers. Here we present TFBS‐Discovery Tool Hub (TDTHub), a web server for quick and intuitive studies of transcriptional regulation in plants. TDTHub uses pre‐computed TFBSs in 40 plant species and allows the choice of two mapping algorithms, providing a higher versatility. Besides the main TFBS enrichment tool, TDTHub includes additional tools to assist in the analysis and visualization of data. In order to demonstrate the effectiveness of TDTHub, we analyzed the transcriptional regulation of the anthocyanin biosynthesis pathway. We also analyzed the transcriptional cascades in response to jasmonate and wounding in Arabidopsis and tomato (Solanum lycopersicum), respectively. In these studies, TDTHub helped to verify the most relevant TF nodes and to propose new ones with a prominent role in these pathways. TDTHub is available at http://acrab.cnb.csic.es/TDTHub/, and it will be periodically upgraded and expanded for new species and gene annotations.

A novel RFLP method for identification of morphologically similar avian Sarcocystis species.

Protozoans of genus Sarcocystis are widespread parasites infecting mammals, birds, and reptiles. Morphology of their sarcocysts is an important criterion for species identification. However, as more and more morphologically similar Sarcocystis species are being found and described, additional methods for their routine diagnostics are needed. We investigated restriction fragment length polymorphism (RFLP) as potential alternative to sequencing data analysis for the identification of Sarcocystis species using birds as an intermediate host. The internal transcribed spacer 1 (ITS1) region sequences of seventeen Sarcocystis species (S. albifronsi, S. anasi, S. calchasi, S. columbae, S. cornixi, S. corvusi, S. cristata, S. halieti, S. falcatula, S. fulicae, S. kutkienae, S. lari, S. lindsayi, S. rileyi, S. turdusi, S.wenzeli, and S. wobeseri) of interest were analysed and five best-fitting endonucleases generating most informative restriction fragments were selected for routine testing. In general, RFLP analyses are always inconclusive as they target very short DNA sequences. However, it can be an irreplaceable technique when fast and cheap identification and discrimination of known species are required, which was our main goal and preliminary results indicate that RFLP could be successfully used when identifying closely related avian Sarcocystis species with just two nucleases.

Sequence to size-based separation using microfluidic electrophoresis for targeted cell-free DNA analysis

The aim of this research is to present a new method to identify and separate target DNA of the same size, in base pairs (bp), into different sizes based on the targeted sequences. This sequence-specific analysis can then be used to evaluate the presence of multiple targeted analytes in a sample without the need for fluorescence detection. This work displays the feasibility of this method using multiple different 150 bp target sequences separated via microfluidic electrophoresis into 230 bp to 330 bp peaks. Using a combination of denaturation, hybridization, ligation, purification, and universal amplification, this represents a simple, robust method for targeted analysis of short DNA sequences. This work shows a limit of detection of 3 pg (∼1.825 x 107 copies) of input DNA using 20 PCR cycles and the ability for the method to be used for short DNA sequences extracted from a plasma sample, most importantly cell-free DNA. Overall, this method has the potential to be used for mutation detection and multiplexed analysis without the need for multiple fluorophores or significant optimization due to varying melting temperatures between PCR primers and can qualitatively evaluate the presence of specific target sequences in a variety of molecular diagnostic applications.

IProm-Zea: A two-layer model to identify plant promoters and their types using convolutional neural network

A promoter is a short DNA sequence near the start codon, responsible for initiating the transcription of a specific gene in the genome. The accurate recognition of promoters is important for achieving a better understanding of transcriptional regulation. Because of their importance in the process of biological transcriptional regulation, there is an urgent need to develop in silico tools to identify promoters and their types in a timely and accurate manner. A number of prediction methods have been developed in this regard; however, almost all of them are merely used for identifying promoters and their strength or sigma types. The TATA box region in TATA promoter influences the post-transcriptional processes; therefore, in the current study, we developed a two-layer predictor called “iProm-Zea” using the convolutional neural network (CNN) for identify TATA and TATA less promoters. The first layer can be used to identify a given DNA sequence as a promoter or non-promoter. The second layer can be used to identify whether the recognized promoter is the TATA promoter. To find an optimal feature encoding scheme and model, we employed four feature encoding schemes on different machine learning and CNN algorithms, and based on the evaluation results, we selected a one-hot encoding scheme and a CNN model for iProm-Zea. The 5-fold cross validation testing results demonstrated that the constructed predictor showed great potential for identifying promoters and classifying them as TATA and TATA less promoters. Furthermore, we performed cross-species analysis of iProm-Zea to evaluate its performance in other species. Moreover, to make it easier for other experimental scientists to obtain the results they need, we established a freely accessible and user-friendly web server at http://nsclbio.jbnu.ac.kr/tools/iProm-Zea/.

PredPromoter-MF(2L): A Novel Approach of Promoter Prediction Based on Multi-source Feature Fusion and Deep Forest.

Promoters short DNA sequences play vital roles in initiating gene transcription. However, it remains a challenge to identify promoters using conventional experiment techniques in a high-throughput manner. To this end, several computational predictors based on machine learning models have been developed, while their performance is unsatisfactory. In this study, we proposed a novel two-layer predictor, called PredPromoter-MF(2L), based on multi-source feature fusion and ensemble learning. PredPromoter-MF(2L) was developed based on various deep features learned by a pre-trained deep learning network model and sequence-derived features. Feature selection based on XGBoost was applied to reduce fused features dimensions, and a cascade deep forest model was trained on the selected feature subset for promoter prediction. The results both fivefold cross-validation and independent test demonstrated that PredPromoter-MF(2L) outperformed state-of-the-art methods.

KmerKeys: a web resource for searching indexed genome assemblies and variants.

K-mers are short DNA sequences that are used for genome sequence analysis. Applications that use k-mers include genome assembly and alignment. However, the wider bioinformatic use of these short sequences has challenges related to the massive scale of genomic sequence data. A single human genome assembly has billions of k-mers. As a result, the computational requirements for analyzing k-mer information is enormous, particularly when involving complete genome assemblies. To address these issues, we developed a new indexing data structure based on a hash table tuned for the lookup of short sequence keys. This web application, referred to as KmerKeys, provides performant, rapid query speeds for cloud computation on genome assemblies. We enable fuzzy as well as exact sequence searches of assemblies. To enable robust and speedy performance, the website implements cache-friendly hash tables, memory mapping and massive parallel processing. Our method employs a scalable and efficient data structure that can be used to jointly index and search a large collection of human genome assembly information. One can include variant databases and their associated metadata such as the gnomAD population variant catalogue. This feature enables the incorporation of future genomic information into sequencing analysis. KmerKeys is freely accessible at https://kmerkeys.dgi-stanford.org.

Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips.

Digital quantification based on counting of individual molecules is a promising approach for different biomedical applications due to its enhanced sensitivity. Here, we present a method for the digital detection of nucleic acids (DNA and RNA) on silicon microchips based on the counting of gold nanoparticles (GNPs) in DNA duplexes by scanning electron microscopy (SEM). Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microchips. Then biotin is revealed by a streptavidin–GNP conjugate followed by the detection of GNPs. Sharp images of each nanoparticle allow the visualization of hybridization results on a single-molecule level. The technique was shown to provide highly sensitive quantification of both short oligonucleotide and long double-strand DNA sequences up to 800 bp. The lowest limit of detection of 0.04 pM was determined for short 19-mer oligonucleotide. The method’s applicability was demonstrated for the multiplex quantification of several β-lactamase genes responsible for the development of bacterial resistance against β-lactam antibiotics. Determination of nucleic acids is effective for both specific DNA in lysates and mRNA in transcripts. The method is also characterized by high selectivity for single-nucleotide polymorphism discrimination. The proposed principle of digital quantification is a perspective for studying the mechanisms of bacterial antibiotic resistance and bacterial response to drugs.

The creeping bentgrass microbiome: Traditional culturing and sequencing results compared with metagenomic techniques

AbstractRecent metagenomic studies have probed the fungal microbiome of intensively managed turfgrasses to better understand the organisms present, which may be beneficial or harmful, but the taxonomic resolution is often limited to the family or genus level. This may relate to the common practice of targeting short ribosomal DNA sequences for estimating fungal abundance and phylogenetic relationships. We collected samples of intensively managed creeping bentgrass (Agrostis stolonifera L.) from Guelph, ON, across two growing seasons and obtained 2,204 foliar epiphytic fungal isolates. Sequencing the entire internal transcribed spacer (ITS) region of 251 representative isolates resolved these to 54 species in 31 genera. A comparison of the taxa identified here versus those reported in five metagenomic studies revealed similarities. However, of the 31 genera we identified by sequencing, 13 genera (42%) were not reported in the metagenomic studies related to intensively managed turfgrass systems. The five metagenomic studies identified an average of 44 genera, with 46% (ranging from 4 to 72%) on average being unique to each study. In addition to revealing genera that were not reported in other studies, full‐length ITS sequencing had the advantage of being able to resolve to the species level. We could resolve 248 of the sequenced isolates to species with an e‐value of 10−50, with three left at the genus level. Until sequencing technologies can yield full‐length ITS sequencing, laborious traditional culturing followed by sequencing of the entire ITS region can give insights into microbiomes not revealed by current metagenomic methods.

Mitogenomic analyses shed new light on phylogenetic relationships within the genus Ruditapes (Bivalvia: Veneridae)

ABSTRACT Ruditapes aspera is a member of the family Veneridae, the venus clams. This species is morphologically similar to other two globally economically important Ruditapes species, R. decussatus and R. philippinarum. Recent phylogenetic studies of Veneridae based on short DNA sequences have suggested that these species may be less closely related than originally thought. Relationships among species of Ruditapes clearly require further critical study. In this article, we show that the mitochondrial (mt) genome of R. aspera exhibits sex-linked heteroplasmy, that is doubly uniparental inheritance (DUI). Mitogenomic comparisons among three Ruditapes species showed that the features and gene arrangements of their mt genomes did not exhibit a high degree of similarity (this agrees with published data on other congeneric species of Veneridae) and that none of these species have a sister-group relationship with either of the other two species. These three species may belong to three different genera, but a fully revised classification will require considerable additional genomic and morphological data from a dense sampling of species and genera. Furthermore, we show that the pattern of relationships in our phylogeny of Ruditapes provides support for the polyphyletic origin of DUI.

Detection of Short DNA Sequences with DNA Nanopores.

Several studies suggest strong correlation between different types of cancer and the relative concentration of short circulating RNA sequences (miRNA). Because of short length and low concentration, miRNA detection is not easy. Standard methods such as RT-PCR require both the standard PCR amplification step and a preliminary additional step of reverse transcription. In this paper, we investigate the use of DNA nanopores as a tool to detect short oligonucleotide sequences at the single molecule level. These nanostructures show two different conformations depending on the presence of DNA analogues of miRNA sequences. By monitoring current across a lipid bilayer, we show that this change of conformation translates to different levels of conductance.

Crystal structure of the RNA cleaving 10-23 DNAzyme captured in a pre-catalytic state

Deoxyribozymes (DNAzymes) are short single-stranded DNA sequences capable of catalyzing chemical reactions such as RNA cleavage, thymine excision, RNA/DNA ligation, and thymine dimer resolution. Among these, RNA cleaving DNAzymes have emerged as the most prominent due to their applications in medicine as potential protein expression knockdown therapeutics and antiviral agents; however, the lack of a structural insight into catalysis hinders further optimization. Here, we report a structure of the RNA cleaving 10-23 DNAzyme, which requires magnesium as a cofactor.

Single-molecule studies of the role of CTP in the action of ParB protein

ParB protein is a component of ParABS system and plays an essential role in plasmid partitioning and bacterial chromosome segregation. It specifically recognizes short parS DNA sequences and spreads to vicinity regions. Recent discoveries indicate that ParB proteins are novel enzymes that bind and hydrolyze cytidine triphosphate (CTP) in their actions, but their working mechanisms are still elusive. We aimed to reveal how the presence of CTP affects the function of Bacillus subtilis ParB (BsParB or Spo0J) protein by utilizing single-molecule flow-stretching assays.

Programmable Macroscopic Self-Assembly of DNA-Decorated Hydrogels.

The precise and predictable formation of double-helical structures from complementary DNA sequences has made DNA an extremely versatile tool for programming self-assembled structures from the nanometer to micrometer scale. While a number of supramolecular interactions have been shown to drive self-assembly of macroscopic building blocks of the millimeter scale, DNA-driven self-assembly of macroscopic objects has not been well-established. In this work, we developed a postpolymerization coupling strategy to conjugate short DNA sequences to polyacrylamide-based hydrogel blocks. We observed sequence-specific self-assembly of DNA-decorated hydrogels with 1-2 mm edges in aqueous solution. Furthermore, selective disassembly of hydrogels upon addition of a DNA strand was demonstrated by exploiting a strand displacement reaction. These results lay the foundation for adaptation of various DNA functions to macroscopic self-assembly, for example, molecular recognition, molecular computation, and chemical catalysis.

A Localized Enantioselective Catalytic Site on Short DNA Sequences and Their Amphiphiles.

A DNA-based artificial metalloenzyme (ArM) consisting of a copper(II) complex of 4,4′-dimethyl-2,2′-bipyridine (dmbipy-Cu) bound to double-stranded DNA (dsDNA) as short as 8 base pairs with only 2 contiguous central pairs (G for guanine and C for cytosine) catalyzes the highly enantioselective Diels–Alder reaction, Michael addition, and Friedel–Crafts alkylation in water. Molecular simulations indicate that these minimal sequences provide a single site where dmbipy-Cu is groove-bound and able to function as an enantioselective catalyst. Enantioselective preference inverts when d-DNA is replaced with l-DNA. When the DNA is conjugated to a hydrophobic tail, the obtained ArMs exhibit enantioselective performance in a methanol–water mixture superior to that of non-amphiphilic dsDNA, and dsDNA-amphiphiles with more complex G•C-rich sequences.

Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features

Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´′- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´′-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.