Multiple Diverse Circoviruses Infect Farm Animals and Are Commonly Found in Human and Chimpanzee Feces

Circoviruses are known to infect birds and pigs and can cause a wide range of severe symptoms with significant economic impact. Using viral metagenomics, we identified circovirus-like DNA sequences and characterized 15 circular viral DNA genomes in stool samples from humans in Pakistan, Nigeria, Tunisia, and the United States and from wild chimpanzees. Distinct genomic features and phylogenetic analysis indicate that some viral genomes were part of a previously unrecognized genus in the Circoviridae family we tentatively named “Cyclovirus” whose genetic diversity is comparable to that of all the known species in the Circovirus genus. Circoviridae detection in the stools of U.S. adults was limited to porcine circoviruses which were also found in most U.S. pork products. To determine whether the divergent cycloviruses found in non-U.S. human stools were of dietary origin, we genetically compared them to the cycloviruses in muscle tissue samples of commonly eaten farm animals in Pakistan and Nigeria. Limited genetic overlap between cycloviruses in human stool samples and local cow, goat, sheep, camel, and chicken meat samples indicated that the majority of the 25 Cyclovirus species identified might be human viruses. We show that the genetic diversity of small circular DNA viral genomes in various mammals, including humans, is significantly larger than previously recognized, and frequent exposure through meat consumption and contact with animal or human feces provides ample opportunities for cyclovirus transmission. Determining the role of cycloviruses, found in 7 to 17% of non-U.S. human stools and 3 to 55% of non-U.S. meat samples tested, in both human and animal diseases is now facilitated by knowledge of their genomes.Animal viruses with small, circular, single-stranded DNA (ssDNA) genomes comprise the Circoviridae family and the Anellovirus genus, while viruses in the Geminiviridae and Nanoviridae families infect plants (3, 25, 34, 37, 40). The genomes of these small viruses without a lipid envelope replicate through a rolling-circle mechanism, possibly sharing a common origin with bacterial plasmids (6), and show high recombination and nucleotide substitution rates (7, 19).The Circoviridae family consists of the Circovirus genus whose member species are currently known to infect only birds and pigs, and the Gyrovirus genus, including a single species, Chicken anemia virus (CAV). Circoviruses infect several avian groups, including parrots, pigeons, gulls, anserids (ducks, geese, and swans), and numerous passerines (ravens, canaries, finches, and starlings) (12, 15, 16, 22, 26, 31, 35, 38, 39). Avian circoviruses have been associated with a variety of symptoms, including developmental abnormalities, lymphoid depletion, and immunosuppression (22, 26, 28, 35, 39). Mammalian circoviruses include only two closely related species, Porcine circovirus 1 and 2 (PCV1 and PCV2, respectively), infecting pigs (21). PCV2 has been associated with porcine circovirus-associated diseases, which can manifest as a systemic disease, respiratory disease complex, enteric disease, porcine dermatitis and nephropathy syndrome or as reproductive problems, causing great losses in the pork industry (1, 29, 32). Circovirus infections are thought to occur mainly through fecal-oral transmission (37).We describe here highly diverse, circovirus-like, circular DNA viral genomes discovered in human and chimpanzee stool samples, and we propose their inclusion in a new genus of the Circoviridae family that we tentatively name “Cyclovirus” pending review by the International Committee on Taxonomy of Viruses (ICTV). Cycloviruses were also found to be prevalent in the muscle tissue of farm animals, such as chickens, cows, sheep, goats, and camels. The cyclovirus species found in human stool samples and in animal meat samples showed limited genetic overlap, suggesting that most of the cycloviruses found in human stool samples are not from consumed animal meat. Rather, these cycloviruses in human stools might cause human enteric infections. The presence of cycloviruses in human stool samples and in farm animal tissue also suggests the potential for frequent cross-species exposure and zoonotic transmissions.     

Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.     

Full Genome Virus Detection in Fecal Samples Using Sensitive Nucleic Acid Preparation,Deep Sequencing,and a Novel Iterative Sequence Classification Algorithm

We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis.     

The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns

Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.     

Analysis of the Gull Fecal Microbial Community Reveals the Dominance of Catellicoccus marimammalium in Relation to Culturable Enterococci

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-β-d-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment.     

Over 50,000 Metagenomically Assembled Draft Genomes for the Human Oral Microbiome Reveal New Taxa

The oral cavity of each person is home to hundreds of bacterial species. While taxa for oral diseases have been studied using culture-based characterization as well as amplicon sequencing, metagenomic and genomic information remains scarce compared to the fecal microbiome. Here, using metagenomic shotgun data for 3346 oral metagenomic samples together with 808 published samples, we obtain 56,213 metagenome-assembled genomes (MAGs), and more than 64% of the 3589 species-level genome bins (SGBs) contain no publicly available genomes. The resulting genome collection is representative of samples around the world and contains many genomes from candidate phyla radiation (CPR) that lack monoculture. Also, it enables the discovery of new taxa such as a genus Candidatus Bgiplasma within the family Acholeplasmataceae. Large-scale metagenomic data from massive samples also allow the assembly of strains from important oral taxa such as Porphyromonas and Neisseria. The oral microbes encode genes that could potentially metabolize drugs. Apart from these findings, a strongly male-enriched Campylobacter species was identified. Oral samples would be more user-friendly collected than fecal samples and have the potential for disease diagnosis. Thus, these data lay down a genomic framework for future inquiries of the human oral microbiome.     

Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors

Background

The genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated.

Results

Three extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans–Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host.

Conclusions

Three extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1321-y) contains supplementary material, which is available to authorized users.     

Viral metagenomics analysis of planktonic viruses in East Lake, Wuhan, China

East Lake (Lake Donghu), located in Wuhan, China, is a typical city freshwater lake that has been experiencing eutrophic conditions and algal blooming during recent years. Marine and fresh water are considered to contain a large number of viruses. However, little is known about their genetic diversity because of the limited techniques for culturing viruses. In this study, we conducted a viral metagenomic analysis using a high-throughput sequencing technique with samples collected from East Lake in Spring, Summer, Autumn, and Winter. The libraries from four samples each generated 234,669, 71,837, 12,820, and 34,236 contigs (> 90 bp each), respectively. The genetic structure of the viral community revealed a high genetic diversity covering 23 viral families, with the majority of contigs homologous to DNA viruses, including members of Myoviridae, Podoviridae, Siphoviridae, Phycodnaviridae, and Microviridae, which infect bacteria or algae, and members of Circoviridae, which infect invertebrates and vertebrates. The highest viral genetic diversity occurred in samples collected in August, then December and June, and the least diversity in March. Most contigs have low-sequence identities with known viruses. PCR detection targeting the conserved sequences of genes (g20, psbA, psbD, and DNApol) of cyanophages further confirmed that there are novel cyanophages in the East Lake. Our viral metagenomic data provide the first preliminary understanding of the virome in one freshwater lake in China and would be helpful for novel virus discovery and the control of algal blooming in the future.     

A Duplex Real-Time PCR Assay for the Simultaneous Detection of Porcine Circovirus 2 and Circovirus 3

Porcine circoviruses (PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.     

Distribution of Plasmids in Distinct Leptospira Pathogenic Species

Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups—pathogens, non-pathogens, and intermediates—based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological significance might contribute to our understanding of biology and infectivity of pathogenic spirochetes.     

Transmission of the yeast mitochondrial genome to progeny: The impact of intergenic sequences

Summary In a previous publication it was shown that the output of yeast mitochondrial loci lacking nearby intergenic sequences (encompassing ori/rep elements) was reduced in crosses to strains with wild-type mtDNAs. In the present work, mitochondrial genomes carrying the intergenic deletions were marked at unlinked, loci by introducing specific antibiotic resistance mutations against erythromycin, oligomycin and paromomycin. These marked genomes were used to follow the output of unlinked regions of the genome from crosses between the intergenic deletion mutants and wild-type strains. Transmission of genetically unlinked markers in coding regions was substantially reduced when an intergenic deletion was present on the same genome. In general the transmission of the antibiotic markers was the same as or slightly higher than the corresponding intergenic marker. These results indicate that the presence of an intergenic deletion in the regions studied impairs the transmission to progeny of a mitochondrial genome as a whole. More specifically, the results suggest that ori/rep sequences, present in the regions that have been deleted, confer a competitive advantage over genomes lacking a full complement of such sequences. These results support the hypothesis that intergenic sequences, and specifically ori/rep elements, have a biological role in the mitochondrial genome. However, because of the exclusive presence of ori/rep sequences in the genus Saccharomyces, it may be that these sequences evolved in (or invaded) the mitochondrial genome relatively late in the evolution of the yeasts. Therefore, in a more general sense, variations in the amount and structure of intergenic sequences in various yeasts may reflect processes that have been of selective advantage in the metabolism of individual mitochondrial DNA in a particular environment and that have not drastically interrupted the respiratory phenotype.     

Characterisation of the Plasmidome within Enterococcus faecalis Isolated from Marginal Periodontitis Patients in Norway

The present study aimed to identify and characterize plasmids in a national collection of oral Enterococcus faecalis (n = 106) isolated from patients with marginal periodontitis. Plasmid replicon typing was performed by multiplex-PCR and sequencing with specific primers for 18 rep-families and 1 unique sequence. Additional plasmid analysis by S1-PFGE was performed for comparison. Totally 120 plasmid replicon amplicons of seven rep-families were identified in 93 E. faecalis strains, e.g. rep9 (prototype pCF10), rep6 (prototype pS86), rep2 (prototype pRE25/pEF1), and rep8 (prototype pAM373). Rep9 was the most predominant rep-family being detected in 81 (76.4%) strains. Forty of these strains were tetracycline resistant and three were erythromycin resistant. Rep6 was the second predominant rep-family being detected in 22 (20.8%) strains. Rep2 was detected in eight (7.5%) strains. All rep2-positive strains were resistant to tetracycline and/or erythromycin and six of them contained Tn916/Tn1545 genes. The rep-positive E. faecalis exhibited divergence in multilocus sequence types (STs). There was a significant correlation between rep9 and ST21, while multiple rep-families appeared in ST40. Totally 145 plasmid bands were identified in 95 E. faecalis strains by S1-PFGE, 59 strains carrying one plasmid, 27 carrying two, five carrying three, three carrying four, and one strain carrying five plasmids. Plasmid sizes varied between 5–150 kbp. There was a significant correlation between the number of plasmids identified by PCR rep-typing and by S1-PFGE. The results indicate that the majority of E. faecalis of marginal periodontitis are likely to be a reservoir for diverse mobile genetic elements and associated antimicrobial resistance determinants.     

The methylomes of six bacteria

Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and C. jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N⁶-methyladenine (^m6A) and N⁴-methylcytosine (^m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases, it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTase genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. Two of these proved active. No attempt was made to detect 5-methylcytosine (^m5C) recognition motifs from the SMRT® sequencing data because this modification produces weaker signals using current methods. However, all predicted ^m6A and ^m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active but also revealing their recognition sequences.     

Diversity of Telomere Palindromic Sequences and Replication Genes among Streptomyces Linear Plasmids

Streptomyces sp. linear plasmids and linear chromosomes usually contain conserved terminal palindromic sequences bound by the conserved telomeric proteins Tap and Tp, encoded by the tap and tpg genes, respectively, as well as plasmid loci required for DNA replication in circular mode when the telomeres are deleted. These consist of iterons and an adjacent rep gene. By using PCR, we found that 8 of 17 newly detected linear plasmids in Streptomyces strains lack typical telomeric tap and tpg sequences. Instead, two novel telomeres in plasmids pRL1 and pRL2 from the eight strains and one conserved telomere in pFRL1 from the other strains were identified, while multiple short palindromes were also found in the plasmids. The complete nucleotide sequence of pRL2 revealed a gene encoding a protein containing two domains, resembling Tap of Streptomyces and a helicase of Thiobacillus, and an adjacent gene encoding a protein similar to Tpg of Streptomyces and a portion of the telomere terminal protein pTP of adenoviruses. No typical iterons-rep loci were found in the three plasmids. These results indicate an unexpected diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids.     

BAC-Pool Sequencing and Analysis of Large Segments of A12 and D12 Homoeologous Chromosomes in Upland Cotton

Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.     

Odintifier - A computational method for identifying insertions of organellar origin from modern and ancient high-throughput sequencing data based on haplotype phasing

Background

Cellular organelles with genomes of their own (e.g. plastids and mitochondria) can pass genetic sequences to other organellar genomes within the cell in many species across the eukaryote phylogeny. The extent of the occurrence of these organellar-derived inserted sequences (odins) is still unknown, but if not accounted for in genomic and phylogenetic studies, they can be a source of error. However, if correctly identified, these inserted sequences can be used for evolutionary and comparative genomic studies. Although such insertions can be detected using various laboratory and bioinformatic strategies, there is currently no straightforward way to apply them as a standard organellar genome assembly on next-generation sequencing data. Furthermore, most current methods for identification of such insertions are unsuitable for use on non-model organisms or ancient DNA datasets.

Results

We present a bioinformatic method that uses phasing algorithms to reconstruct both source and inserted organelle sequences. The method was tested in different shotgun and organellar-enriched DNA high-throughput sequencing (HTS) datasets from ancient and modern samples. Specifically, we used datasets from lions (Panthera leo ssp. and Panthera leo leo) to characterize insertions from mitochondrial origin, and from common grapevine (Vitis vinifera) and bugle (Ajuga reptans) to characterize insertions derived from plastid genomes. Comparison of the results against other available organelle genome assembly methods demonstrated that our new method provides an improvement in the sequence assembly.

Conclusion

Using datasets from a wide range of species and different levels of complexity we showed that our novel bioinformatic method based on phasing algorithms can be used to achieve the next two goals: i) reference-guided assembly of chloroplast/mitochondrial genomes from HTS data and ii) identification and simultaneous assembly of odins. This method represents the first application of haplotype phasing for automatic detection of odins and reference-based organellar genome assembly.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0682-1) contains supplementary material, which is available to authorized users.     

Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers

In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined in sewage spiked samples and environmental waters using different group-specific and host-specific PCR assays. Bacteroidales DNA recovery ranged from 59 to 71% for aliquots of supernatant collected after the elution step. The recovery of human-specific Bacteroidales DNA from sewage spiked samples was 54% in the elution step. An additional 1-7% Bacteroidales DNA was recovered after the immunomagnetic separation step, while recovery from the pellet left after the immunomagnetic separation of protozoa parasites was substantially lower. Comparison of Bacteroidales 16S rRNA gene sequences from elution and immunomagnetic separation discarded samples indicated that the distribution of clones was not statistically different, suggesting that there were no recovery biases introduced by these steps. Human- and cow-specific Bacteroidales and fecal indicator bacteria (i.e., enterococci,) were also detected in the discarded fractions of environmental samples collected from different geographic locations. Overall, the results of this study demonstrated the potential application of leftover sample fractions that are currently discarded for the PCR detection of fecal bacterial indicators and molecular source tracking.     

Bifidobacteria in Feces and Environmental Waters

Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays showed that Bifidobacterium adolescentis, B. bifidum, B. dentium, and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve, B. infantis, and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 bifidobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured bifidobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Bifidobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host specificity. Using species-specific assays, bifidobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of bifidobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable.     

Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR

Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4 × 10⁴ target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10³ copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R² ≥ 0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters.     

The Intestinal Eukaryotic Virome in Healthy and Diarrhoeic Neonatal Piglets

Neonatal porcine diarrhoea of uncertain aetiology has been reported from a number of European countries. The aim of the present study was to use viral metagenomics to examine a potential viral involvement in this diarrhoea and to describe the intestinal virome with focus on eukaryotic viruses. Samples from the distal jejunum of 50 diarrhoeic and 19 healthy piglets from 10 affected herds were analysed. The viral fraction of the samples was isolated and nucleic acids (RNA and DNA fractions) were subjected to sequence independent amplification. Samples from diarrhoeic piglets from the same herds were pooled whereas samples from healthy piglets were analysed individually. In total, 29 clinical samples, plus two negative controls and one positive control consisting of a mock metagenome were sequenced using the Ion Torrent platform. The resulting sequence data was subjected to taxonomic classification using Kraken, Diamond and HMMER. In the healthy specimens, eight different mammalian virus families were detected (Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Parvoviridae, Picornaviridae, and Reoviridae) compared to four in the pooled diarrhoeic samples (Anelloviridae, Circoviridae, Picornaviridae, and Reoviridae). It was not possible to associate a particular virus family with the investigated diarrhoea. In conclusion, this study does not support the hypothesis that the investigated diarrhoea was caused by known mammalian viruses. The results do, however, indicate that known mammalian viruses were present in the intestine as early as 24–48 hours after birth, indicating immediate infection post-partum or possibly transplacental infection.