Targeted DNA sequencing: rapid identification of DNA clones by sequencing DNA using mixed oligodeoxynucleotide probes as primers

A rapid identification method involving targeted DNA sequencing of genomic or cDNA clones using mixed (degenerate) probes as primers is described. The strategy involves the use of the same mixed probes for sequencing the clone of interest as they are used for screening the DNA libraries. Probes containing up to 512 mixes do not interfere in priming and yield completely faithful replication of the template DNA.     

Cellular Lipid and Fatty Acid Compositions of Burkholderia pseudomallei Strains Isolated from Human and Environment in Viet Nam

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and ?2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1: 1. Since snake venom phospholipase A₂ digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16: 0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16: 0), 2-hydroxy hexadecenoic (2-OH-C16: 1), 2-hydroxy octadecenoic (2-OH-C18: 1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14: 0) and 3-hydroxy palmitic (3-OH-C16: 0) acids. There were significant differences in the concentration of hexadecenoic (C16: 1), methylene hexadecanoic (C17CPA), octadecenoic (C18: 1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.     

Distribution and abundance of marine turtles in the Socialist Republic of Viet Nam

To establish baseline data on the distribution, abundance and threats to marine turtles in Viet Nam we conducted surveys with local fishers, community members and provincial Ministry of Fisheries staff from 17 of Viet Nam’s 29 coastal provinces. These data indicate that five species of marine turtle reside in Viet Nam’s waters (loggerhead, olive ridley, leatherback, green and hawksbill turtles), and four species nest on Viet Nam’s beaches (all of the above except the loggerhead turtle). It is evident from these data that significant declines have occurred in both foraging and nesting populations of all five marine turtle species found in Viet Nam. The greatest current threats to marine turtle populations in Viet Nam are habitat degradation, the accidental and opportunistic of turtles capture by fishers and the direct take of nesting females and their eggs. Successful conservation efforts have been made in recent years through collaboration between international Non Government Organisations and several Vietnamese Government Ministries. Continued success of these projects and the development and implementation of marine conservation policy will depend upon building awareness among Government employees, fishers and the general public about marine turtle biology, ecology, and the need to protect them.     

Primates in northern Viet Nam: A review of the ecology and conservation status of extant species,with notes on pleistocene localities

We discuss the distribution of living and fossil primates in northern Viet Nam, integrate new localities into the known ranges, and address the ecological parameters and status of extant primate species. In addition, we interpolate fossil specimens from Pleistocene cave sites into the discussion of current ranges. This approach provides a unique glimpse into the past ranges of extant species in a tropical setting, which is unavailable for most regions of the world. Finally, we review the effects of ecocide and other anthropogenic threats to nonhuman primates in Viet Nam.     

PCR amplification of microalgal DNA for sequencing and species identification: studies on fixatives and algal growth stages

Cultured strains and individually isolated dinoflagellate cells from field samples were preserved in different fixatives to find a method of cell preservation that could provide DNA template in PCR reactions and preserve cell morphology for microscopic studies. Lugol’s solution and various ethanol concentrations all showed shortcomings, whereas an initial formalin preservation step followed by storage in 100% methanol fulfilled both demands. Cells could be stored up to 1 year and still provide functional DNA template for positive PCR reactions. The amplified fragment was approximately 700 bp of the D1/D2 region of the LSU rDNA, which is to our knowledge significantly longer than the low-molecular-weight DNA typically reported from formalin preserved samples. By cloning and sequencing the PCR product and subsequently aligning the sequences with previously sequenced fragments of the same or similar species, we confirmed that no base pair alteration had taken place during the time that the cells were fixed and frozen. In another experiment it was demonstrated that the growth phase of cultured Alexandrium minutum did not have any influence on the result of PCR reactions. This was true for extracted DNA from cultures and for direct PCR with a small number of disrupted cells. Phenol/chlorophorm/isoamylalcohol extraction proved to be an unpredictable method for DNA extraction, whereas direct PCR on isolated cells was more reliable. Extracted DNA purified with a commercial DNA cleaning kit always rendered a positive PCR. The environmental condition for cells to be used as DNA template in PCR is discussed.     

Rapid identification of multi-strain HBV infection in patient by high-throughput DNA sequencing

Hepatitis B is a well-known risk factor for the development of liver cancer and is closely associated with patient morbidity and mortality. Viral mutants and variants have the potential to evade immune response and prolong infection, and thus it is crucial to develop a methodology for the rapid identification of multi-strain hepatitis infections in patients. Here we describe a method based on selective region amplification of viral genome and deep sequencing, which may be used for rapid identification of multi-strain hepatitis B virus (HBV) infection in patients. The method works even with significantly low amounts of patients’ serum samples, where the wet-lab procedures take about 1.5 days, followed by a quick bioinformatic analysis to reveal the final results. Our method can potentially be applied to the rapid and reliable identification of multi-strain HBV infection and help improve treatment regiments.     

Comparing whole‐genome shotgun sequencing and DNA metabarcoding approaches for species identification and quantification of pollen species mixtures

Molecular identification of mixed‐species pollen samples has a range of applications in various fields of research. To date, such molecular identification has primarily been carried out via amplicon sequencing, but whole‐genome shotgun (WGS) sequencing of pollen DNA has potential advantages, including (1) more genetic information per sample and (2) the potential for better quantitative matching. In this study, we tested the performance of WGS sequencing methodology and publicly available reference sequences in identifying species and quantifying their relative abundance in pollen mock communities. Using mock communities previously analyzed with DNA metabarcoding, we sequenced approximately 200Mbp for each sample using Illumina HiSeq and MiSeq. Taxonomic identifications were based on the Kraken k‐mer identification method with reference libraries constructed from full‐genome and short read archive data from the NCBI database. We found WGS to be a reliable method for taxonomic identification of pollen with near 100% identification of species in mixtures but generating higher rates of false positives (reads not identified to the correct taxon at the required taxonomic level) relative to rbcL and ITS2 amplicon sequencing. For quantification of relative species abundance, WGS data provided a stronger correlation between pollen grain proportion and sequence read proportion, but diverged more from a 1:1 relationship, likely due to the higher rate of false positives. Currently, a limitation of WGS‐based pollen identification is the lack of representation of plant diversity in publicly available genome databases. As databases improve and costs drop, we expect that eventually genomics methods will become the methods of choice for species identification and quantification of mixed‐species pollen samples.     

Extraction of microsporidial DNA from modified trichrome-stained clinical slides and subsequent species identification using PCR sequencing

Molecular techniques involving polymerase chain reaction (PCR) and sequencing provide a relatively simple and objective means of identifying microsporidia to species level. We modified previously described methods of DNA extraction and PCR conditions for identification of microsporidia from museum slides, clinical specimens and environmental samples and successfully identified Vittaforma corneae in 11 out of 13 cases of microsporidial infection from used trichrome-stained slides of corneal scrapings from HIV-negative patients with keratoconjunctivitis.     

Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia

DNA coding for the 16S rRNA of an intracellular bacterium was directly amplified from lysed cells of a host amoebae using the polymerase chain reaction and primers specific for eubacteria. The amoebae had been used to recover an uncultured bacterium observed in the sputum of a patient with pneumonia. The amplified DNA was sequenced directly and compared with published 16S rRNA sequences. The analysis revealed that the intracellular bacterium is a member of the genus Legionella and that it is different from species, including L. pneumophila, for which 16S ribosomal RNA sequence data are available.     

Strain and species identification by restriction fragment length polymorphisms in the ribosomal DNA repeat of Candida species

Restriction fragment length polymorphisms in the ribosomal DNA (rDNA) have been shown to be a useful criterion for distinguishing among various isolates of Candida albicans. In a sample of 12 clinical isolates, we found six different classes based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA. Some of the classes appeared to be heterozygous at the rDNA locus. Similar digestion of other Candida species showed that each could be identified on the basis of its restriction patterns. Since these are highly reiterated genes, the differences were apparent on ethidium bromide-stained gels; Southern transfers were not necessary. EcoRI restriction maps of the rDNA of C. albicans, C. stellatoidea, C. tropicalis, and C. guilliermondii were determined.     

Identification of fish species by an egg identification key: an alternative to the incubation of eggs and rearing of larvae for species recognition

Comparison of the results of two methods for species recognition, an egg identification key and rearing of larvae, showed only small qualitative differences in the identification of fish species present in a stretch of the River Elbe, Germany. Both the identification key and rearing methods complement each other as methods of identification, and the selection of which method to be used should depend primarily on the aim of the investigation.     

Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance

Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.     

Identifying live bird markets with the potential to act as reservoirs of avian influenza A (H5N1) virus: a survey in northern Viet Nam and Cambodia

Wet markets are common in many parts of the world and may promote the emergence, spread and maintenance of livestock pathogens, including zoonoses. A survey was conducted in order to assess the potential of Vietnamese and Cambodian live bird markets (LBMs) to sustain circulation of highly pathogenic avian influenza virus subtype H5N1 (HPAIV H5N1). Thirty Vietnamese and 8 Cambodian LBMs were visited, and structured interviews were conducted with the market managers and 561 Vietnamese and 84 Cambodian traders. Multivariate and cluster analysis were used to construct a typology of traders based on their poultry management practices. As a result of those practices and large poultry surplus (unsold poultry reoffered for sale the following day), some poultry traders were shown to promote conditions favorable for perpetuating HPAIV H5N1 in LBMs. More than 80% of these traders operated in LBMs located in the most densely populated areas, Ha Noi and Phnom Penh. The profiles of sellers operating at a given LBM could be reliably predicted using basic information about the location and type of market. Consequently, LBMs with the largest combination of risk factors for becoming virus reservoirs could be easily identified, potentially allowing control strategies to be appropriately targeted. These findings are of particular relevance to resource-scarce settings with extensively developed LBM systems, commonly found in South-East Asia.     

Human infections of fish-borne trematodes in Vietnam: prevalence and molecular specific identification at an endemic commune in Nam Dinh province

The prevalence of fish-borne trematodes in humans and their molecular identification was investigated in the Rang Dong commune of Nam Dinh province, Vietnam, between January 2009 and December 2010. A total of 405 people in this commune were interviewed on the habit of eating raw fish and all of their stool samples were collected using the Kato-Katz technique for examination of the presence of fish-borne trematodes. The worms (and eggs) were first morphologically examined, counted, described and identified, then the representative isolates were subjected for molecular species confirmation. A total of 385 adult flukes collected from 10 patients were morphologically identified to species and defined as Clonorchis sinensis (14.58%) in Opisthorchiidae family, Haplorchis taichui (32.29%), Haplorchis pumilio (52.08%) and Centrocestus formosanus (1.04%) in Heterophyidae family. A high rate (77.8%) of the interviewees was found to have the habit of eating raw fish. This habit was attributed to the high infection rate of fish-borne trematode in humans (22.72%; OR = 2.486). The infection rate of fish-borne trematodes in males was higher (29.3%) than that in females (16.0%) and increased by age, reaching the highest in the patients aged 40–59 years (28.2–28.7%). The infection intensity of fish-borne trematode was found light (336 EPG). Adult flukes were collected from a group of the patients with the highest intensity of infection and subjected to molecular and phylogenetic analysis using a portion (326 bp) of mitochondrial cox1. Phylogenetic tree inferred from cox1 sequences using sequence data for 34 isolates of opisthorchid, heterophyid, fasciolid, paragonimid, schistosomid trematodes and taeniid cestodes revealed that they are distinct groups. The newly collected with the known clonorchid and heterophyid isolates form the well defined taxonomic groups, respectively, confirming that C. sinensis and Haplorchis spp. (H. pumilio and H. taichui) were among the collected samples.     

All-Food-Seq (AFS): a quantifiable screen for species in biological samples by deep DNA sequencing

Background

DNA-based methods like PCR efficiently identify and quantify the taxon composition of complex biological materials, but are limited to detecting species targeted by the choice of the primer assay. We show here how untargeted deep sequencing of foodstuff total genomic DNA, followed by bioinformatic analysis of sequence reads, facilitates highly accurate identification of species from all kingdoms of life, at the same time enabling quantitative measurement of the main ingredients and detection of unanticipated food components.

Results

Sequence data simulation and real-case Illumina sequencing of DNA from reference sausages composed of mammalian (pig, cow, horse, sheep) and avian (chicken, turkey) species are able to quantify material correctly at the 1% discrimination level via a read counting approach. An additional metagenomic step facilitates identification of traces from animal, plant and microbial DNA including unexpected species, which is prospectively important for the detection of allergens and pathogens.

Conclusions

Our data suggest that deep sequencing of total genomic DNA from samples of heterogeneous taxon composition promises to be a valuable screening tool for reference species identification and quantification in biosurveillance applications like food testing, potentially alleviating some of the problems in taxon representation and quantification associated with targeted PCR-based approaches.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-639) contains supplementary material, which is available to authorized users.     

Quantitative analysis of DNA in sea urchin eggs and subcellular distribution of DNA in the eggs

An improved procedure for analyzing DNA in animal eggs which contain a great deal of other chromogenic materials has been developed. As a result the following DNA contents were estimated: 3.59 ± 0.39 pg DNA/egg for H. pulcherrimus, 5.06 ± 0.30 pg DNA/egg for Ps. depressus, and 3.78 ± 0.33 pg DNA/egg for A. crassispina. Egg nuclei contain the haploid level of DNA. Most of the cytoplasmic DNA was found to be associated with the mitochondria (more than 90%) and only a small portion was detected in the yolk granule fraction.     

Identification of the amino acid residues of the amino terminus of vimentin responsible for DNA binding by enzymatic and chemical sequencing and analysis by MALDI-TOF

The amino acid residues responsible for stable binding of nucleic acids by the intermediate filament (IF) subunit protein vimentin were identified by a combination of enyzmatic and chemical ladder sequencing of photo-cross-linked vimentin-oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry. Three tryptic peptides of vimentin (vim(28)(-)(35), vim(36)(-)(49), and vim(50)(-)(63)) were found to be cross-linked to oligo(dG.BrdU)(12). dG.3'-FITC. From a methodological standpoint, it was necessary to remove the bulk of the bound oligonucleotide by digestion with nuclease P1 to get reproducible spectra for most of the peptides studied. Additionally, removal of the phosphate group of the residually bound dUMP or modification of the amino terminus of the peptide-oligonucleotide complexes with dimethylaminoazobenzene isothiocyanate dramatically improved the quality of the MALDI-TOF spectra obtained, particularly for the vim(28)(-)(35) peptide. A single Tyr residue within each of these peptides (Tyr(29), Tyr(37), and Tyr(52)) was unequivocally demonstrated to be the unique site of cross-linking in each peptide. These three Tyr residues are contained within the two beta-ladder DNA-binding wings proposed for the middle of the vimentin non-alpha-helical head domain. The experimental approach described should be generally applicable to the study of protein-nucleic acid interactions and is currently being employed to characterize the DNA-binding sites of several other IF subunit proteins.