Exploring bacterial diversity via a curated and searchable snapshot of archived DNA sequences

The open sharing of genomic data provides an incredibly rich resource for the study of bacterial evolution and function and even anthropogenic activities such as the widespread use of antimicrobials. However, these data consist of genomes assembled with different tools and levels of quality checking, and of large volumes of completely unprocessed raw sequence data. In both cases, considerable computational effort is required before biological questions can be addressed. Here, we assembled and characterised 661,405 bacterial genomes retrieved from the European Nucleotide Archive (ENA) in November of 2018 using a uniform standardised approach. Of these, 311,006 did not previously have an assembly. We produced a searchable COmpact Bit-sliced Signature (COBS) index, facilitating the easy interrogation of the entire dataset for a specific sequence (e.g., gene, mutation, or plasmid). Additional MinHash and pp-sketch indices support genome-wide comparisons and estimations of genomic distance. Combined, this resource will allow data to be easily subset and searched, phylogenetic relationships between genomes to be quickly elucidated, and hypotheses rapidly generated and tested. We believe that this combination of uniform processing and variety of search/filter functionalities will make this a resource of very wide utility. In terms of diversity within the data, a breakdown of the 639,981 high-quality genomes emphasised the uneven species composition of the ENA/public databases, with just 20 of the total 2,336 species making up 90% of the genomes. The overrepresented species tend to be acute/common human pathogens, aligning with research priorities at different levels from individual interests to funding bodies and national and global public health agencies.

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Exploring bacterial diversity via a curated and searchable snapshot of archived DNA sequences

10.1101/2021.03.02.433662 ◽

2021 ◽

Author(s):

Grace A. Blackwell ◽

Martin Hunt ◽

Kerri M. Malone ◽

Leandro Lima ◽

Gal Horesh ◽

...

Keyword(s):

Bacterial Diversity ◽

Dna Sequences ◽

Anthropogenic Activities ◽

Research Priorities ◽

Specific Gene ◽

Human Pathogens ◽

Public Health Agencies ◽

European Nucleotide Archive ◽

National Public Health ◽

And Function

ABSTRACTThe open sharing of genomic data provides an incredibly rich resource for the study of bacterial evolution and function, and even anthropogenic activities such as the widespread use of antimicrobials. Whilst these archives are rich in data, considerable processing is required before biological questions can be addressed. Here, we assembled and characterised 661,405 bacterial genomes using a uniform standardised approach, retrieved from the European Nucleotide Archive (ENA) in November of 2018. A searchable COBS index has been produced, facilitating the easy interrogation of the entire dataset for a specific gene or mutation. Additional MinHash and pp-sketch indices support genome-wide comparisons and estimations of genomic distance. An analysis on this scale revealed the uneven species composition in the ENA/public databases, with just 20 of the total 2,336 species making up 90% of the genomes. The over-represented species tend to be acute/common human pathogens. This aligns with research priorities at different levels from individuals with targeted but focused research questions, areas of focus for the funding bodies or national public health agencies, to those identified globally as priority pathogens by the WHO for their resistance to front and last line antimicrobials. Understanding the actual and potential biases in bacterial diversity depicted in this snapshot, and hence within the data being submitted to the public sequencing archives, is essential if we are to target and fill gaps in our understanding of the bacterial kingdom.

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Characterization of squalene synthase gene from Gymnema sylvestre R. Br.

Beni-Suef University Journal of Basic and Applied Sciences ◽

10.1186/s43088-020-00094-4 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Kuldeepsingh A. Kalariya ◽

Ram Prasnna Meena ◽

Lipi Poojara ◽

Deepa Shahi ◽

Sandip Patel

Keyword(s):

Dna Sequences ◽

Genomic Dna ◽

Competitive Inhibition ◽

Sequence Data ◽

Homology Model ◽

Squalene Synthase ◽

Gymnema Sylvestre ◽

Gardenia Jasminoides ◽

Ramachandran Plots ◽

Flanking Regions

Abstract Background Squalene synthase (SQS) is a rate-limiting enzyme necessary to produce pentacyclic triterpenes in plants. It is an important enzyme producing squalene molecules required to run steroidal and triterpenoid biosynthesis pathways working in competitive inhibition mode. Reports are available on information pertaining to SQS gene in several plants, but detailed information on SQS gene in Gymnema sylvestre R. Br. is not available. G. sylvestre is a priceless rare vine of central eco-region known for its medicinally important triterpenoids. Our work aims to characterize the GS-SQS gene in this high-value medicinal plant. Results Coding DNA sequences (CDS) with 1245 bp length representing GS-SQS gene predicted from transcriptome data in G. sylvestre was used for further characterization. The SWISS protein structure modeled for the GS-SQS amino acid sequence data had MolProbity Score of 1.44 and the Clash Score 3.86. The quality estimates and statistical score of Ramachandran plots analysis indicated that the homology model was reliable. For full-length amplification of the gene, primers designed from flanking regions of CDS encoding GS-SQS were used to get amplification against genomic DNA as template which resulted in approximately 6.2-kb sized single-band product. The sequencing of this product through NGS was carried out generating 2.32 Gb data and 3347 number of scaffolds with N50 value of 457 bp. These scaffolds were compared to identify similarity with other SQS genes as well as the GS-SQSs of the transcriptome. Scaffold_3347 representing the GS-SQS gene harbored two introns of 101 and 164 bp size. Both these intronic regions were validated by primers designed from adjoining outside regions of the introns on the scaffold representing GS-SQS gene. The amplification took place when the template was genomic DNA and failed when the template was cDNA confirmed the presence of two introns in GS-SQS gene in Gymnema sylvestre R. Br. Conclusion This study shows GS-SQS gene was very closely related to Coffea arabica and Gardenia jasminoides and this gene harbored two introns of 101 and 164 bp size.

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Islands of complex DNA are widespread in Drosophila centric heterochromatin.

Genetics ◽

10.1093/genetics/141.1.283 ◽

1995 ◽

Vol 141 (1) ◽

pp. 283-303

Author(s):

M H Le ◽

D Duricka ◽

G H Karpen

Keyword(s):

Dna Sequences ◽

Structure And Function ◽

Southern Analysis ◽

Cloning And Sequencing ◽

Pulsed Field ◽

Drosophila Heterochromatin ◽

And Function ◽

Heterochromatin Structure ◽

Derivatives Of ◽

Eukaryotic Genomes

Abstract Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three "islands" of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed.

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Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽

10.1186/s13568-019-0890-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Neeraja Punde ◽

Jennifer Kooken ◽

Dagmar Leary ◽

Patricia M. Legler ◽

Evelina Angov

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Codon Usage ◽

Dna Sequences ◽

Structural Integrity ◽

Host Cells ◽

Loss Of Function ◽

Species Specific ◽

And Function ◽

The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

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Molecular evolution of homologous gene sequences in germline-limited and somatic chromosomes of Acricotopus

Genome ◽

10.1139/g04-026 ◽

2004 ◽

Vol 47 (4) ◽

pp. 732-741 ◽

Cited By ~ 2

Author(s):

Wolfgang Staiber

Keyword(s):

Molecular Evolution ◽

Dna Sequences ◽

Sequence Data ◽

Structural Evolution ◽

5S Rdna ◽

Oligonucleotide Primer ◽

Homologous Gene ◽

Gene Sequences ◽

Nucleotide Substitutions ◽

Degenerate Oligonucleotide

The origin of germline-limited chromosomes (Ks) as descendants of somatic chromosomes (Ss) and their structural evolution was recently elucidated in the chironomid Acricotopus. The Ks consist of large S-homologous sections and of heterochromatic segments containing germline-specific, highly repetitive DNA sequences. Less is known about the molecular evolution and features of the sequences in the S-homologous K sections. More information about this was received by comparing homologous gene sequences of Ks and Ss. Genes for 5.8S, 18S, 28S, and 5S ribosomal RNA were choosen for the comparison and therefore isolated first by PCR from somatic DNA of Acricotopus and sequenced. Specific K DNA was collected by microdissection of monopolar moving K complements from differential gonial mitoses and was then amplified by degenerate oligonucleotide primer (DOP)-PCR. With the sequence data of the somatic rDNAs, the homologous 5.8S and 5S rDNA sequences were isolated by PCR from the DOP-PCR sequence pool of the Ks. In addition, a number of K DOP-PCR sequences were directly cloned and analysed. One K clone contained a section of a putative N-acetyltransferase gene. Compared with its homolog from the Ss, the sequence exhibited few nucleotide substitutions (99.2% sequence identity). The same was true for the 5.8S and 5S sequences from Ss and Ks (97.5%100% identity). This supports the idea that the S-homologous K sequences may be conserved and do not evolve independently from their somatic homologs. Possible mechanisms effecting such conservation of S-derived sequences in the Ks are discussed.Key words: microdissection, DOP-PCR, germline-limited chromosomes, molecular evolution.

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A species-specific satellite DNA from the entomopathogenic nematode Heterorhabditis indicus

Genome ◽

10.1139/g98-005 ◽

1998 ◽

Vol 41 (2) ◽

pp. 148-153 ◽

Cited By ~ 8

Author(s):

Monique Abadon ◽

Eric Grenier ◽

Christian Laumond ◽

Pierre Abad

Keyword(s):

Dna Sequence ◽

Satellite Dna ◽

Tandem Repeats ◽

Sequence Data ◽

Entomopathogenic Nematode ◽

Consensus Sequence ◽

Repeated Sequence ◽

Nucleotide Sequence Analysis ◽

Specific Sequence ◽

Species Specific

An AluI satellite DNA family has been cloned from the entomopathogenic nematode Heterorhabditis indicus. This repeated sequence appears to be an unusually abundant satellite DNA, since it constitutes about 45% of the H. indicus genome. The consensus sequence is 174 nucleotides long and has an A + T content of 56%, with the presence of direct and inverted repeat clusters. DNA sequence data reveal that monomers are quite homogeneous. Such homogeneity suggests that some mechanism is acting to maintain the homogeneity of this satellite DNA, despite its abundance, or that this repeated sequence could have appeared recently in the genome of H. indicus. Hybridization analysis of genomic DNAs from different Heterorhabditis species shows that this satellite DNA sequence is specific to the H. indicus genome. Considering the species specificity and the high copy number of this AluI satellite DNA sequence, it could provide a rapid and powerful tool for identifying H. indicus strains.Key words: AluI repeated DNA, tandem repeats, species-specific sequence, nucleotide sequence analysis.

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Predicting Chromosome Flexibility from the Genomic Sequence Based on Deep Learning Neural Networks

Current Bioinformatics ◽

10.2174/1574893616666210827095829 ◽

2021 ◽

Vol 16 ◽

Author(s):

Jinghao Peng ◽

Jiajie Peng ◽

Haiyin Piao ◽

Zhang Luo ◽

Kelin Xia ◽

...

Keyword(s):

Deep Learning ◽

High Performance ◽

Genomic Sequence ◽

Sequence Data ◽

Function Analysis ◽

Double Helix ◽

Gm12878 Cell ◽

Genomic Sequence Analysis ◽

And Function ◽

Nuclear Processes

Background: The open and accessible regions of the chromosome are more likely to be bound by transcription factors which are important for nuclear processes and biological functions. Studying the change of chromosome flexibility can help to discover and analyze disease markers and improve the efficiency of clinical diagnosis. Current methods for predicting chromosome flexibility based on Hi-C data include the flexibility-rigidity index (FRI) and the Gaussian network model (GNM), which have been proposed to characterize chromosome flexibility. However, these methods require the chromosome structure data based on 3D biological experiments, which is time-consuming and expensive. Objective: Generally, the folding and curling of the double helix sequence of DNA have a great impact on chromosome flexibility and function. Motivated by the success of genomic sequence analysis in biomolecular function analysis, we hope to propose a method to predict chromosome flexibility only based on genomic sequence data. Method: We propose a new method (named "DeepCFP") using deep learning models to predict chromosome flexibility based on only genomic sequence features. The model has been tested in the GM12878 cell line. Results: The maximum accuracy of our model has reached 91%. The performance of DeepCFP is close to FRI and GNM. Conclusion: The DeepCFP can achieve high performance only based on genomic sequence.

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Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening

International Journal of Molecular Sciences ◽

10.3390/ijms19113664 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3664 ◽

Cited By ~ 1

Author(s):

Ali Munawar ◽

Steven Beelen ◽

Ahmad Munawar ◽

Eveline Lescrinier ◽

Sergei Strelkov

Keyword(s):

Zika Virus ◽

Dissociation Constants ◽

Global Public Health ◽

Human Pathogens ◽

Allosteric Site ◽

Replication Complex ◽

X Ray ◽

X Ray Crystallography ◽

Fragment Screening ◽

Atomic Precision

The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015–2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (KD) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.

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SHI7 Is a Self-Learning Pipeline for Multipurpose Short-Read DNA Quality Control

mSystems ◽

10.1128/msystems.00202-17 ◽

2018 ◽

Vol 3 (3) ◽

Cited By ~ 15

Author(s):

Gabriel A. Al-Ghalith ◽

Benjamin Hillmann ◽

Kaiwei Ang ◽

Robin Shields-Cutler ◽

Dan Knights

Keyword(s):

Quality Control ◽

Dna Sequences ◽

Sequence Data ◽

Background Knowledge ◽

Sequencing Technology ◽

Data Set ◽

Short Read ◽

Dna Quality ◽

Public Data ◽

User Friendly

ABSTRACT Next-generation sequencing technology is of great importance for many biological disciplines; however, due to technical and biological limitations, the short DNA sequences produced by modern sequencers require numerous quality control (QC) measures to reduce errors, remove technical contaminants, or merge paired-end reads together into longer or higher-quality contigs. Many tools for each step exist, but choosing the appropriate methods and usage parameters can be challenging because the parameterization of each step depends on the particularities of the sequencing technology used, the type of samples being analyzed, and the stochasticity of the instrumentation and sample preparation. Furthermore, end users may not know all of the relevant information about how their data were generated, such as the expected overlap for paired-end sequences or type of adaptors used to make informed choices. This increasing complexity and nuance demand a pipeline that combines existing steps together in a user-friendly way and, when possible, learns reasonable quality parameters from the data automatically. We propose a user-friendly quality control pipeline called SHI7 (canonically pronounced “shizen”), which aims to simplify quality control of short-read data for the end user by predicting presence and/or type of common sequencing adaptors, what quality scores to trim, whether the data set is shotgun or amplicon sequencing, whether reads are paired end or single end, and whether pairs are stitchable, including the expected amount of pair overlap. We hope that SHI7 will make it easier for all researchers, expert and novice alike, to follow reasonable practices for short-read data quality control. IMPORTANCE Quality control of high-throughput DNA sequencing data is an important but sometimes laborious task requiring background knowledge of the sequencing protocol used (such as adaptor type, sequencing technology, insert size/stitchability, paired-endedness, etc.). Quality control protocols typically require applying this background knowledge to selecting and executing numerous quality control steps with the appropriate parameters, which is especially difficult when working with public data or data from collaborators who use different protocols. We have created a streamlined quality control pipeline intended to substantially simplify the process of DNA quality control from raw machine output files to actionable sequence data. In contrast to other methods, our proposed pipeline is easy to install and use and attempts to learn the necessary parameters from the data automatically with a single command.

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SiteOut: an online tool to design binding site-free DNA sequences

10.1101/029645 ◽

2015 ◽

Cited By ~ 1

Author(s):

Javier Estrada ◽

Teresa Ruiz-Herrero ◽

Clarissa Scholes ◽

Zeba Wunderlich ◽

Angela DePace

Keyword(s):

Binding Site ◽

Dna Sequences ◽

Binding Sites ◽

Regulatory Proteins ◽

Biological Processes ◽

Major Goal ◽

Specific Sequence ◽

Online Tool ◽

Protein Binding Sites ◽

Regulatory Dna

DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/

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