Census-based rapid and accurate metagenome taxonomic profiling

Background

Understanding the taxonomic composition of a sample, whether from patient, food or environment, is important to several types of studies including pathogen diagnostics, epidemiological studies, biodiversity analysis and food quality regulation. With the decreasing costs of sequencing, metagenomic data is quickly becoming the preferred typed of data for such analysis.

Results

Rapidly defining the taxonomic composition (both taxonomic profile and relative frequency) in a metagenomic sequence dataset is challenging because the task of mapping millions of sequence reads from a metagenomic study to a non-redundant nucleotide database such as the NCBI non-redundant nucleotide database (nt) is a computationally intensive task. We have developed a robust subsampling-based algorithm implemented in a tool called CensuScope meant to take a ‘sneak peak’ into the population distribution and estimate taxonomic composition as if a census was taken of the metagenomic landscape. CensuScope is a rapid and accurate metagenome taxonomic profiling tool that randomly extracts a small number of reads (based on user input) and maps them to NCBI’s nt database. This process is repeated multiple times to ascertain the taxonomic composition that is found in majority of the iterations, thereby providing a robust estimate of the population and measures of the accuracy for the results.

Conclusion

CensuScope can be run on a laptop or on a high-performance computer. Based on our analysis we are able to provide some recommendations in terms of the number of sequence reads to analyze and the number of iterations to use. For example, to quantify taxonomic groups present in the sample at a level of 1% or higher a subsampling size of 250 random reads with 50 iterations yields a statistical power of >99%. Windows and UNIX versions of CensuScope are available for download at https://hive.biochemistry.gwu.edu/dna.cgi?cmd=censuscope. CensuScope is also available through the High-performance Integrated Virtual Environment (HIVE) and can be used in conjunction with other HIVE analysis and visualization tools.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-918) contains supplementary material, which is available to authorized users.     

High levels of genetic variability and differentiation in hilsa shad, Tenualosa ilisha (Clupeidae, Clupeiformes) populations revealed by PCR-RFLP analysis of the mitochondrial DNA D-loop region

The hilsa shad, Tenualosa ilisha (Clupeidae, Clupeiformes) is an important anadromous clupeid species from the Western division of the Indo-Pacific region. It constitutes the largest single fishable species in Bangladesh. Information on genetic variability and population structure is very important for both management and conservation purposes. Past reports on the population structure of T. ilisha involving morphometric, allozyme and RAPD analyses are contradictory. We examined genetic variability and divergence in two riverine (the Jamuna and the Meghna), two estuarine (Kuakata and Sundarbans) and one marine (Cox's Bazar) populations of T. ilisha by applying PCR-RFLP analysis of the mtDNA D-loop region. The amplified PCR products were restricted with four restriction enzymes namely, XbaI, EcoRI, EcoRV, and HaeIII. High levels of haplotype and gene diversity within and significant differentiations among, populations of T. ilisha were observed in this study. Significant F(ST) values indicated differentiation among the river, estuary and marine populations. The UPGMA dendrogram based on genetic distance resulted in two major clusters, although, these were subsequently divided into three, corresponding to the riverine, estuarine and marine populations. The study underlines the usefulness of RFLP of mtDNA D-loop region as molecular markers, and detected at least two differentiated populations of T. ilisha in Bangladesh waters.     

Conformational behavior of α-d-mannopyranosyl-(1→6)-α,β-d-mannose complexed with two mannose-binding plant lectins, Allium sativam agglutinin I and concanavalin A, using NMR and molecular modeling techniques

Herein, we report the intrinsic conformational preferences of α-d-Manp-(1→6)-α,β-d-Manp, (1) in the free state and as two (ASAI and ConA) lectin-bound forms. NMR spectroscopy and molecular dynamics techniques are used as 3D-structural determination tools. In free form disaccharide 1 displays a fair amount of conformational freedom, with one major (?/ψ 95 ± 30°/195 ± 20°) and one minor (95 ± 30°/70 ± 20°) conformations around the glycosidic linkage and around the ω angle, both the gg and gt rotamers are almost equally populated. This is a first report of a three-dimensional structure of 1 bound with ASAI. Both lectins recognize a major ?/ψ 95 ± 30°/200 ± 30° conformer with the ligand showing more flexibility in the binding site of ConA. Comparison of the mode of binding of the two lectins explains the differences in observed specificities.     

Purification and characterization of a novel caffeine oxidase from Alcaligenes species

Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.     

Perturbations in the catfish immune responses by arsenic: organ and cell specific effects

The present study was an attempt to elucidate the effect of non-lethal arsenic (As) exposure (1/10 LC50) on different immunologically important organs and cells of a catfish. Clarias batrachus L. were exposed to arsenic trioxide for different time intervals, which resulted in significant, time-dependent changes in total head kidney and splenic leucocyte count (p<0.05) and reduction in the organosomatic indices (p<0.05) of these two important immunocompetent organs. Routine histological studies revealed arsenic induced changes in the cellular composition of head kidney and spleen. Arsenic also induced time-dependent and tissue-specific alterations in T and B cell functioning in catfish. When checked for its effects on macrophages, it was noted that arsenic interfered with bacterial phagocytosis. Furthermore, arsenic affected the general immune status of C. batrachus and rendered the fish immunocompromised and susceptible to pathogens.     

Torrefaction: a sustainable method for transforming of agri-wastes to high energy density solids (biocoal)

Reviews in Environmental Science and Bio/Technology - The depletion of fossil fuel reserves and greenhouse gas emissions led to limit the use of fossil fuels, including natural gas, coal, or...     

Prevalence of Diarrhea-Associated Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Animal Hosts

In order to assess the health risk associated with a given source of fecal contamination using bacterial source tracking (BST), it is important to know the occurrence of potential pathogens as a function of host. Escherichia coli isolates (n = 593) from the feces of diverse animals were screened for various virulence genes: stx₁ and stx₂ (Shiga toxin-producing E. coli [STEC]), eae and EAF (enteropathogenic E. coli [EPEC]), STh, STp, and LT (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). Eleven hosts were positive for only the eae (10.11%) gene, representing atypical EPEC, while two hosts were positive for both eae and EAF (1.3%), representing typical EPEC. stx₁, stx₂, or both stx₁ and stx₂ were present in 1 (0.1%,) 10 (5.56%), and 2 (1.51%) hosts, respectively, and confirmed as non-O157 by using a E. coli O157 rfb (rfb_O157) TaqMan assay. STh and STp were carried by 2 hosts (2.33%) and 1 host (0.33%), respectively, while none of the hosts were positive for LT and ipaH. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 221 unique fingerprints with a Shannon diversity index of 2.67. Multivariate analysis of variance revealed that majority of the isolates clustered according to the year of sampling. The higher prevalence of atypical EPEC and non-O157 STEC observed in different animal hosts indicates that they can be a reservoir of these pathogens with the potential to contaminate surface water and impact human health. Therefore, we suggest that E. coli from these sources must be included while constructing known source fingerprint libraries for tracking purposes. However, the observed genetic diversity and temporal variation need to be considered since these factors can influence the accuracy of BST results.     

Defective chromatin recruitment and retention of NHEJ core components in human tumor cells expressing a Cyclin E fragment

Exposure to genotoxic agents, such as ionizing radiation (IR), produces double-strand breaks, repaired predominantly in mammalian cells by non-homologous end-joining (NHEJ). Ku70 was identified as an interacting partner of a proteolytic Cyclin E (CycE) fragment, p18CycE. p18CycE endogenous generation during IR-induced apoptosis in leukemic cells and its stable expression in epithelial tumor cells sensitized to IR. γH2AX IR-induced foci (IRIFs) and comet assays indicated ineffective NHEJ DNA repair in p18CycE-expressing cells. DNA pull-down and chromatin recruitment assays revealed that retention of NHEJ factors to double-strand breaks, but not recruitment, was diminished. Similarly, IRIFs of phosphorylated T2609 and S2056-DNA-PKcs and its target S1778-53BP1 were greatly decreased in p18CycE-expressing cells. As a result, DNA-PKcs chromatin association was also increased. 53BP1 IRIFs were suppressed when p18CycE was generated in leukemic cells and in epithelial cells stably expressing p18CycE. Ataxia telangiectasia mutated was activated but not its 53BP1 and MDC1 targets. These data indicate a profound influence of p18CycE on NHEJ through its interference with DNA-PKcs conformation and/or dimerization, which is required for effective DNA repair, making the p18CycE-expressing cells more IR sensitive. These studies provide unique mechanistic insights into NHEJ misregulation in human tumor cells, in which defects in NHEJ core components are rare.     

Brevibacillus laterosporus strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam, India

A bacterial strain designated as BPM3 isolated from mud of a natural hot water spring of Nambar Wild Life Sanctuary, Assam, India, strongly inhibited growth of phytopathogenic fungi (Fusarium oxysporum f. sp. ciceri, F. semitectum, Magnaporthe grisea and Rhizoctonia oryzae) and gram-positive bacterium (Staphylococcus aureus). The maximum growth and antagonistic activity was recorded at 30 °C, pH 8.5 when starch and peptone were amended as carbon and nitrogen sources, respectively. In greenhouse experiment, this bacterium (BPM3) suppressed blast disease of rice by 30-67% and protected the weight loss by 35-56.5%. The maximum disease protection (67%) and weight loss protection (56.5%) were recorded when the bacterium was applied before 2 days of the pathogen inoculation. Antifungal and antibacterial compounds were isolated from the bacterium which also inhibited the growth of these targeted pathogens. The compounds were purified and on spectroscopic analysis of a purified fraction having R_f 0.22 which showed strong antifungal and antibacterial activity indicated the presence of C-H, carbonyl group, dimethyl group, -CH₂ and methyl group. The bacterium was characterized by morphological, biochemical and molecular approaches and confirmed that the strain BPM3 is Brevibacillus laterosporus.