Comparative and joint analysis of two metagenomic datasets from a biogas fermenter obtained by 454-pyrosequencing

Biogas production from renewable resources is attracting increased attention as an alternative energy source due to the limited availability of traditional fossil fuels. Many countries are promoting the use of alternative energy sources for sustainable energy production. In this study, a metagenome from a production-scale biogas fermenter was analysed employing Roche's GS FLX Titanium technology and compared to a previous dataset obtained from the same community DNA sample that was sequenced on the GS FLX platform. Taxonomic profiling based on 16S rRNA-specific sequences and an Environmental Gene Tag (EGT) analysis employing CARMA demonstrated that both approaches benefit from the longer read lengths obtained on the Titanium platform. Results confirmed Clostridia as the most prevalent taxonomic class, whereas species of the order Methanomicrobiales are dominant among methanogenic Archaea. However, the analyses also identified additional taxa that were missed by the previous study, including members of the genera Streptococcus, Acetivibrio, Garciella, Tissierella, and Gelria, which might also play a role in the fermentation process leading to the formation of methane. Taking advantage of the CARMA feature to correlate taxonomic information of sequences with their assigned functions, it appeared that Firmicutes, followed by Bacteroidetes and Proteobacteria, dominate within the functional context of polysaccharide degradation whereas Methanomicrobiales represent the most abundant taxonomic group responsible for methane production. Clostridia is the most important class involved in the reductive CoA pathway (Wood-Ljungdahl pathway) that is characteristic for acetogenesis. Based on binning of 16S rRNA-specific sequences allocated to the dominant genus Methanoculleus, it could be shown that this genus is represented by several different species. Phylogenetic analysis of these sequences placed them in close proximity to the hydrogenotrophic methanogen Methanoculleus bourgensis. While rarefaction analyses still indicate incomplete coverage, examination of the GS FLX Titanium dataset resulted in the identification of additional genera and functional elements, providing a far more complete coverage of the community involved in anaerobic fermentative pathways leading to methane formation.     

A framework for developing and validating taxon‐specific primers for specimen identification from environmental DNA

Taxon‐specific DNA tests are applied to many ecological and management questions, increasingly using environmental DNA (eDNA). eDNA facilitates noninvasive ecological studies but introduces additional risks of bias and error. For effective application, PCR primers must be developed for each taxon and validated in each system. We outline a nine step framework for the development and validation of taxon‐specific primers for eDNA analysis in ecological studies, involving reference database construction, phylogenetic evaluation of the target gene, primer design, primer evaluation in silico, and laboratory evaluation of primer specificity, sensitivity and utility. Our framework makes possible a rigorous evaluation of likely sources of error. The first five steps can be conducted relatively rapidly and (where reference DNA sequences are available) require minimal laboratory resources, enabling assessment of primer suitability before investing in further work. Steps six to eight require more costly laboratory analyses but are essential to evaluate risks of false‐positive and false‐negative results, while step 9 relates to field implementation. As an example, we have developed and evaluated primers to specifically amplify part of the mitochondrial ND2 gene from Australian bandicoots. If adopted during the early stages of primer development, our framework will facilitate large‐scale implementation of well‐designed DNA tests to detect specific wildlife from eDNA samples. This will provide researchers and managers with an understanding of the strengths and limitations of their data and the conclusions that can be drawn from them.     

Hybridization ddRAD‐sequencing for population genomics of nonmodel plants using highly degraded historical specimen DNA

Species’ responses at the genetic level are key to understanding the long‐term consequences of anthropogenic global change. Herbaria document such responses, and, with contemporary sampling, provide high‐resolution time‐series of plant evolutionary change. Characterizing genetic diversity is straightforward for model species with small genomes and a reference sequence. For nonmodel species—with small or large genomes—diversity is traditionally assessed using restriction‐enzyme‐based sequencing. However, age‐related DNA damage and fragmentation preclude the use of this approach for ancient herbarium DNA. Here, we combine reduced‐representation sequencing and hybridization‐capture to overcome this challenge and efficiently compare contemporary and historical specimens. Specifically, we describe how homemade DNA baits can be produced from reduced‐representation libraries of fresh samples, and used to efficiently enrich historical libraries for the same fraction of the genome to produce compatible sets of sequence data from both types of material. Applying this approach to both Arabidopsis thaliana and the nonmodel plant Cardamine bulbifera, we discovered polymorphisms de novo in an unbiased, reference‐free manner. We show that the recovered genetic variation recapitulates known genetic diversity in A. thaliana, and recovers geographical origin in both species and over time, independent of bait diversity. Hence, our method enables fast, cost‐efficient, large‐scale integration of contemporary and historical specimens for assessment of genome‐wide genetic trends over time, independent of genome size and presence of a reference genome.     

Penalized classification for optimal statistical selection of markers from high-throughput genotyping: application in sheep breeds

The identification of individuals’ breed of origin has several practical applications in livestock and is useful in different biological contexts such as conservation genetics, breeding and authentication of animal products. In this paper, penalized multinomial regression was applied to identify the minimum number of single nucleotide polymorphisms (SNPs) from high-throughput genotyping data for individual assignment to dairy sheep breeds reared in Sicily. The combined use of penalized multinomial regression and stability selection reduced the number of SNPs required to 48. A final validation step on an independent population was carried out obtaining 100% correctly classified individuals. The results using independent analysis, such as admixture, F_st, principal component analysis and random forest, confirmed the ability of these methods in selecting distinctive markers. The identified SNPs may constitute a starting point for the development of a SNP based identification test as a tool for breed assignment and traceability of animal products.     

In vitro selection of optimal DNA substrates for ligation by a water-soluble carbodiimide

We have used in vitro selection to investigate the sequence requirements for efficient template-directed ligation of oligonucleotides at 0°C using a water-soluble carbodiimide as condensing agent. We find that only 2 by at each side of the ligation junction are needed. We also studied chemical ligation of substrate ensembles that we have previously selected as optimal for ligation by RNA ligase or by DNA ligase. As anticipated, we find that substrates selected with DNA ligase ligate efficiently with a chemical ligating agent, and vice versa. Substrates selected using RNA ligase are not ligated by the chemical condensing agent and vice versa. The implications of these results for prebiotic chemistry are discussed.Correspondence to: Leslie E. Orgel 1444     

The application of CRISPR‐Cas for single species identification from environmental DNA

We report the first application of CRISPR‐Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR‐based techniques to detect a given species from eDNA samples, posing a logistical challenge for on‐site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR‐Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof‐of‐concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.     

Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection

BackgroundThe broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.

Methodology/Principal Findings

We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.

Conclusions and Significance

We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.     

Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA

Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67 bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.     

Molecular identification of fine roots of trees from the Alps: reliable and fast DNA extraction and PCR-RFLP analyses of plastid DNA

Fine roots of trees are intensively used as indicators to assess soil alterations, e.g. those owing to atmospheric inputs of acidifying substances, but their identification to species with morphological criteria is difficult. In this study, we established molecular techniques in order to identify fine roots of the 30 most common tree species of the Alps. We developed a protocol for efficient isolation of DNA from fine roots with extraction of DNA in the presence of polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP). The trnL (UAA) intron of plastid DNA was used as a marker for fine root identification. We amplified and sequenced this intron with plant universal primers. The size of the sequences ranged from 444 to 672 bp. A synoptic key for species identification was designed on the basis of restriction fragment patterns predicted from sequence data. Using the restriction enzyme TaqI as key enzyme, and where necessary HinfI, RsaI and CfoI, 16 taxa, including Picea abies, Larix decidua, Abies alba, and Fagus sylvatica, the dominant tree species of the Alpine region could be identified by agarose gel electrophoresis of restriction fragments. Fourteen taxa could be identified to the genus level, among them Quercus, Salix and Populus species. In a field study, conducted in a 20 x 30 m plot of a mixed forest with five tree species, fine roots of 43 out of 46 samples were identified and their distributions were mapped. These results demonstrate the utility of our DNA extraction method and of the trnL intron for the identification of fine tree roots.     

非损伤采样提取DNA技术在猫科动物调查中的应用

<正>猫科动物广泛分布于世界各地(Johnson et al.,2006),全球现存37种猫科动物,其中25种已被国际自然保护联盟(IUCN)列为濒危和易危,86%以上的种群数量处于下降或未知状态(IUCN Red List,2011)。我国有13种猫科动物,占世界种类的35%(王应祥,2003),其中属于国家一级保护的有4种,二级保护的有8种,然而,对这些珍稀物种的生态学研究并不充分(高耀亭,     

The application of DNA sequence data for the identification of benthic nematodes from the North Sea

Nematodes or roundworms represent one of the most diverse and dominant taxon in marine benthic habitats. Whereas a morphological identification of many species is challenging, the application of molecular markers represents a promising approach for species discrimination and identification. In this study, we used an integrative taxonomic approach, combining both molecular and morphological methods, to characterize nematodes of distinct sex and ontogenetic stages from three sampling sites of the North Sea. Morphospecies were discriminated after first visual determination, followed by a molecular analysis of the nuclear 28S rDNA: D2–D3 marker. By linking each sequence to a morphological voucher, discordant morphological identification was subjected to a so-called reverse taxonomic approach. Molecular operational taxonomic units (MOTUs) and morphospecies were compared for all of the three sampling sites to assess concordance of methodology. In total, 32 MOTUs and 26 morphospecies were assigned, of which 12 taxa were identified as described species. Both approaches showed high concordance in taxon assignment (84.4 %) except for a cluster comprising various Sabatieria species. Our study revealed the high potential of the analyzed fragment as a useful molecular marker for the identification of the North Sea nematodes and highlighted the applicability of this combined taxonomic approach in general.     

The application of random amplified polymorphic DNA for sandfly species identification

Abstract. We have applied the recently developed Random Amplified Polymorphic DNA (RAPD) method to produce species-specific, DNA profiles for two sympatric, Venezuelan sandfly species, thought to be the vectors responsible for recent outbreaks of cutaneous and mucocutaneous leishmaniasis in the Andean State of Tachira. Moreover, within the profile, it was possible to identify a diagnostic DNA band for Lu.youngi of 0.32 kb. Results showed that the size of this diagnostic DNA band remained constant and did not vary with sex or geographical distribution.     

DNA barcoding of brown Parmeliae (Parmeliaceae) species: a molecular approach for accurate specimen identification,emphasizing species in Greenland

Warming of Arctic and alpine regions has a substantial impact on high-altitude/-latitude ecosystems. Shifting biomes due to climate change may lead to adjustments in species distributions and potential extinctions. Therefore, detailed monitoring is requisite to assess biologically meaningful shifts in community composition and species distributions. Some Arctic-alpine lichens have been shown to be particularly sensitive to climatic shifts associated with global change. However, accurate identification of lichenized fungal species remains challenging and may limit the effective use of lichens in climate change research. Given the inherent difficulties in accurate identification of lichenized fungi and the potential value of efficient identifications for bio-monitoring research, we investigated the utility of DNA barcode identification of the 13 brown Parmeliae (Ascomycota) species occurring in Greenland. For these species, we assessed monophyly and genetic distances using the nuclear ribosomal internal transcribed spacer region (ITS), the standard DNA barcode for fungi. We also compared intraspecific distance values to a proposed intra-interspecific threshold value for Parmeliaceae to identify nominal taxa potentially masking previously unrecognized diversity. Our results indicated that the 13 brown Parmeliae species occurring in Greenland can be successfully discriminated using the ITS region. All phenotypically circumscribed species were recovered as well-supported, monophyletic clades. Furthermore, our data supported a barcode gap among congeners for all brown Parmeliae species investigated here. However, high intraspecific genetic distances suggest the potential for previously unrecognized species-lineages in at least five species: Melanelia agnata, M. hepatizon, Montanelia disjuncta, M. panniformis, and M. tominii. Our research facilitates effective, long-term bio-monitoring of climate change in Greenland using lichens by providing accurate molecular identification of brown Parmeliae specimens.     

Co‐amplification of cytochrome b and D‐loop mtDNA fragments for the identification of degraded DNA samples

We propose a simple and effective approach to simultaneously co‐amplify both cytochrome b and D‐loop fragments to evaluate DNA preservation and to monitor possible contaminations in the analysis of degraded animal DNA samples. We have applied this approach to over 200 ancient salmon samples and 25 ancient whale DNA samples, clearly demonstrating its multiple benefits for analysis of degraded DNA samples, and the ease in which co‐amplification can be optimized for different taxa. This simple, cost‐efficient and genomic DNA‐saving approach can be used routinely in the analysis of minute and degraded DNA samples in wildlife forensics, food inspection, conservation biology and ancient faunal remains.     

Evidence for balancing selection from nucleotide sequence analyses of human G6PD

Glucose-6-phosphate dehydrogenase (G6PD) mutations that result in reduced enzyme activity have been implicated in malarial resistance and constitute one of the best examples of selection in the human genome. In the present study, we characterize the nucleotide diversity across a 5.2-kb region of G6PD in a sample of 160 Africans and 56 non-Africans, to determine how selection has shaped patterns of DNA variation at this gene. Our global sample of enzymatically normal B alleles and A, A-, and Med alleles with reduced enzyme activities reveals many previously uncharacterized silent-site polymorphisms. In comparison with the absence of amino acid divergence between human and chimpanzee G6PD sequences, we find that the number of G6PD amino acid polymorphisms in human populations is significantly high. Unlike many other G6PD-activity alleles with reduced activity, we find that the age of the A variant, which is common in Africa, may not be consistent with the recent emergence of severe malaria and therefore may have originally had a historically different adaptive function. Overall, our observations strongly support previous genotype-phenotype association studies that proposed that balancing selection maintains G6PD deficiencies within human populations. The present study demonstrates that nucleotide sequence analyses can reveal signatures of both historical and recent selection in the genome and may elucidate the impact that infectious disease has had during human evolution.     

应用DNA指纹图谱技术鉴定整肠生菌株BL63516的研究

目的建立应用DNA指纹图谱技术鉴定微生态制剂——整肠生菌株BL63516的方法,提高菌种鉴别水平。方法应用RAPD（随机扩增多态性）方法,采用50条随机引物对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,选择多态性好、重复性好、稳定性强的随机引物,对BL63516与其他地衣芽胞杆菌进行区分。结果发现选用引物$87或$88分别对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,BL63516菌株扩增的DNA片段的大小、数量均与其他地衣芽胞杆菌有明显差异。结论此方法具有可重复性,方便、快速和准确的优势,可用于微生态制剂整肠生菌株的鉴别。