Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Primer extension capture: targeted sequence retrieval from heavily degraded DNA sources.

ABSTRACT: We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

SUBMITTER: Briggs AW

PROVIDER: S-EPMC3150061 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Primer extension capture: targeted sequence retrieval from heavily degraded DNA sources.

Briggs Adrian W AW Good Jeffrey M JM Green Richard E RE Krause Johannes J Maricic Tomislav T Stenzel Udo U Pääbo Svante S

Journal of visualized experiments : JoVE 20090903 31

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximatel ...[more]

PMID: 19730410

Similar Datasets

Primer initiation and extension by T7 DNA primase.

Project description:T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc-binding domain (ZBD) connected by an unstructured linker. The two domains are required to initiate the synthesis of the diribonucleotide pppAC and its extension into a functional primer pppACCC (de novo synthesis), as well as for the extension of exogenous AC diribonucleotides into an ACCC primer (extension synthesis). To explore the mechanism underlying the RPD and ZBD interactions, we have changed the length of the linker between them. Wild-type T7 DNA primase is 10-fold superior in de novo synthesis compared to T7 DNA primase having a shorter linker. However, the primase having the shorter linker exhibits a two-fold enhancement in its extension synthesis. T7 DNA primase does not catalyze extension synthesis by a ZBD of one subunit acting on a RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose a mechanism for primer initiation and extension based on these findings.

| S-EPMC1462978 | biostudies-literature

Microarrays for genotyping human group a rotavirus by multiplex capture and type-specific primer extension.

Project description:Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.

| S-EPMC262494 | biostudies-literature

Enhanced virome sequencing using targeted sequence capture.

Project description:Metagenomic shotgun sequencing (MSS) is an important tool for characterizing viral populations. It is culture independent, requires no a priori knowledge of the viruses in the sample, and may provide useful genomic information. However, MSS can lack sensitivity and may yield insufficient data for detailed analysis. We have created a targeted sequence capture panel, ViroCap, designed to enrich nucleic acid from DNA and RNA viruses from 34 families that infect vertebrate hosts. A computational approach condensed ∼1 billion bp of viral reference sequence into <200 million bp of unique, representative sequence suitable for targeted sequence capture. We compared the effectiveness of detecting viruses in standard MSS versus MSS following targeted sequence capture. First, we analyzed two sets of samples, one derived from samples submitted to a diagnostic virology laboratory and one derived from samples collected in a study of fever in children. We detected 14 and 18 viruses in the two sets, comprising 19 genera from 10 families, with dramatic enhancement of genome representation following capture enrichment. The median fold-increases in percentage viral reads post-capture were 674 and 296. Median breadth of coverage increased from 2.1% to 83.2% post-capture in the first set and from 2.0% to 75.6% in the second set. Next, we analyzed samples containing a set of diverse anellovirus sequences and demonstrated that ViroCap could be used to detect viral sequences with up to 58% variation from the references used to select capture probes. ViroCap substantially enhances MSS for a comprehensive set of viruses and has utility for research and clinical applications.

| S-EPMC4665012 | biostudies-literature

Macroalgal browsing on a heavily degraded, urbanized equatorial reef system.

Project description:The removal of macroalgal biomass is critical to the health of coral reef ecosystems. Previous studies on relatively intact reefs with diverse and abundant fish communities have quantified rapid removal of macroalgae by herbivorous fishes, yet how these findings relate to degraded reef systems where fish diversity and abundance are markedly lower and algal biomass substantially higher, is unclear. We surveyed roving herbivorous fish communities and quantified their capacity to remove the dominant macroalga Sargassum ilicifolium on seven reefs in Singapore; a heavily degraded urbanized reef system. The diversity and abundance of herbivorous fishes was extremely low, with eight species and a mean abundance ~1.1 individuals 60 m-2 recorded across reefs. Consumption of S. ilicifolium varied with distance from Singapore's main port with consumption being 3- to 17-fold higher on reefs furthest from the port (Pulau Satumu: 4.18 g h-1; Kusu Island: 2.38 g h-1) than reefs closer to the port (0.35-0.78 g h-1). Video observations revealed a single species, Siganus virgatus, was almost solely responsible for removing S. ilicifolium biomass, accounting for 83% of the mass-standardized bites. Despite low herbivore diversity and intense urbanization, macroalgal removal by fishes on some Singaporean reefs was directly comparable to rates reported for other inshore Indo-Pacific reefs.

| S-EPMC5567118 | biostudies-literature

Single-nucleotide primer extension assay for detection and sequence typing of "Dehalococcoides" spp.

Project description:A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus "Dehalococcoides". The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.

| S-EPMC2223236 | biostudies-literature

RNA Primer Extension Hinders DNA Synthesis by <i>Escherichia coli</i> Mutagenic DNA Polymerase IV.

Project description:In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB's fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability.

| S-EPMC5331060 | biostudies-literature

Synthesis of N3'-P5'-linked phosphoramidate DNA by nonenzymatic template-directed primer extension.

Project description:A fast and accurate pathway for nonenzymatic RNA replication would simplify models for the emergence of the RNA world from the prebiotic chemistry of the early earth. However, numerous difficulties stand in the way of an experimental demonstration of effective nonenzymatic RNA replication. To gain insight into the necessary properties of potentially self-replicating informational polymers, we have studied several model systems based on amino-sugar nucleotides. Here we describe the synthesis of N3'-P5'-linked phosphoramidate DNA (3'-NP-DNA) by the template-directed polymerization of activated 3'-amino-2',3'-dideoxyribonucleotides. 3'-NP-DNA is an interesting model because of its very RNA-like A-type duplex conformation and because activated 3'-amino-2',3'-dideoxyribonucleotides are much more reactive than the corresponding activated ribonucleotides. In contrast to our previous studies with 2'-amino-2',3'-dideoxyribonucleotides (for which G and C but not A and T exhibit efficient template copying), we have found that all four canonical 3'-amino-2',3'-dideoxyribonucleotides (G, C, A, and T) polymerize efficiently on RNA templates. RNA templates are generally superior to DNA templates, and oligo-ribo-T templates are superior to oligo-ribo-U templates, which are the least efficient of the RNA homopolymer templates. We have also found that activation of 3'-aminonucleotides with 2-methylimidazole results in a ca. 10-fold higher polymerization rate relative to activation with imidazole, an observation that parallels earlier findings with ribonucleotides. We discuss the implications of our experiments for the possibility of self-replication in the 3'-NP-DNA and RNA systems.

| S-EPMC3548433 | biostudies-literature

Molecular basis for DNA double-strand break annealing and primer extension by an NHEJ DNA polymerase.

Project description:Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break (DSB) repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5'-3' direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3' overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3' overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3' ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

| S-EPMC3898472 | biostudies-literature

Optimizing genome editing strategy by primer-extension-mediated sequencing

Project description:High specificity of e\ngineered nucleases ensures precise genome editing. Couple methods were developed to identify off-target sites of CRISPR/Cas9, but hardly any high-throughput sequencing method can unequivocally determine their targeting efficiencies. Here we describe a comprehensive method, primer-extension-mediated sequencing (PEM-seq), which could sensitively detect CRISPR/Cas9 off-target sites as well as assess their editing efficiency by quantifying DNA interference events at on-target sites. Demonstrated by PEM-seq, we generated a high-fidelity Cas9 variant FeCas9 that possesses similar targeting ability as the wild-type while with extremely low off-target activities. Moreover, we provided further evidences for the broader range of xCas9 protospacer adjacent sequence. We also found the AcrIIA4 inhibitor could inhibit both on- and off-target activities of SpCas9, but it suppressed SpCas9 cleavage at the off-target loci not so efficiently as at the on-target sites. Finally, we believe PEM-seq is applicable to optimizing genome editing strategy for clinical purpose or creating animal model.

2019-04-03 | GSE116231 | GEO

Capture MRE-seq for methylation analysis of highly degraded DNA samples

Project description:Understanding the impact of DNA methylation within different disease contexts often requires accurate assessment of these modifications in a genome-wide fashion. Frequently, patient-derived tissue stored in long-term hospital tissue banks have been preserved using formalin-fixation paraffin-embedding (FFPE). While these samples can comprise valuable resources for studying disease, the fixation process ultimately compromises the DNA’s integrity and leads to degradation. Degraded DNA can complicate CpG methylome profiling using traditional techniques, particularly when performing methylation sensitive restriction enzyme sequencing (MRE-seq), yielding high backgrounds and resulting in lowered library complexity. Here, we provide results using our new MRE-seq protocol (Capture MRE-seq), tailored to preserving unmethylated CpG information when using samples with highly degraded DNA. The results using Capture MRE-seq correlate well (0.92) with traditional MRE-seq calls when profiling non-degraded samples, and can recover unmethylated regions in highly degraded samples when traditional MRE-seq fails, which we validate using bisulfite sequencing-based data (WGBS) as well as methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq).

2023-03-01 | GSE155968 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data