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Many HIV-1 envelope-reactive antibodies shortly after HIV-1 transmission may arise from crow-reactive memory B cells previously stimulated by non-HIV-1 host or microbial antigens, The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non–HIV-1 antigens.

Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

Unbiased pyrosequencing detected an astrovirus after conventional methods failed to identify the causative agent., Encephalitis is a major cause of death worldwide. Although >100 pathogens have been identified as causative agents, the pathogen is not determined for up to 75% of cases. This diagnostic failure impedes effective treatment and underscores the need for better tools and new approaches for detecting novel pathogens or determining new manifestations of known pathogens. Although astroviruses are commonly associated with gastroenteritis, they have not been associated with central nervous system disease. Using unbiased pyrosequencing, we detected an astrovirus as the causative agent for encephalitis in a 15-year-old boy with agammaglobulinemia; several laboratories had failed to identify the agent. Our findings expand the spectrum of causative agents associated with encephalitis and highlight unbiased molecular technology as a valuable tool for differential diagnosis of unexplained disease.

Characterization of arboviruses at the interface of pristine habitats and anthropogenic landscapes is crucial to comprehensive emergent disease surveillance and forecasting efforts. In context of a surveillance campaign in and around a West African rainforest, particles morphologically consistent with rhabdoviruses were identified in cell cultures infected with homogenates of trapped mosquitoes. RNA recovered from these cultures was used to derive the first complete genome sequence of a rhabdovirus isolated from Culex decens mosquitoes in Côte d'Ivoire, tentatively named Moussa virus (MOUV). MOUV shows the classical genome organization of rhabdoviruses, with five open reading frames (ORF) in a linear order. However, sequences show only limited conservation (12-33% identity at amino acid level), and ORF2 and ORF3 have no significant similarity to sequences deposited in GenBank. Phylogenetic analysis indicates a potential new species with distant relationship to Tupaia and Tibrogargan virus.

Objectives: K103N, the most common nonnucleoside reverse transcriptase inhibitor (NNRTI)–resistant mutation in patients with transmitted resistance and in patients receiving a failing NNRTIcontaining regimen, is fully susceptible to the new NNRTI, etravirine. Therefore, we sought to determine how often NNRTI-resistant mutations other than K103N occur as minority variants in plasma samples for which standard genotypic resistance testing detects K103N alone. Methods: We performed ultradeep pyrosequencing (UDPS; 454 Life Sciences a Roche Company, Branford, CT) of plasma virus samples from 13 treatment-naive and 20 NNRTI-experienced patients in whom standard genotypic resistance testing revealed K103N but no other major NNRTI-resistance mutations. Results: Samples from 0 of 13 treatment-naive patients vs. 7 of 20 patients failing an NNRTI-containing regimen had minority variants with major etravirine-associated NNRTI-resistant mutations (P = 0.03, Fisher exact test): Y181C (7.0%), Y181C (3.6%) + G190A (3.2%), L100I (14%), L100I (32%) + 190A (5.4%), K101E (3.8%) + G190A (4.9%), K101E (4.0%) + G190S (4.8%), and G190S (3.1%). Conclusions: In treatment-naive patients, UDPS did not detect additional major NNRTI-resistant mutations suggesting that etravirine may be effective in patients with transmitted K103N. In NNRTI-experienced patients, UDPS often detected additional major NNRTI-resistant mutations suggesting that etravirine may not be fully active in patients with acquired K103N.

The genome sequencing of all major food and bioenergy crops is of critical importance in the race to improve crop production to meet the future food and energy security needs of the world. Next generation sequencing technologies have brought about great improvements in sequencing throughput and cost, but do not yet allow for de novo sequencing of large repetitive genomes as found in most crop plants. We present a strategy that combines cutting edge next generation sequencing with “old school” genomics resources and allows rapid cost-effective sequencing of plant genomes.

Background. Minor (i.e.

The human distal gut harbours a vast ensemble of microbes (the microbiota) that provide important metabolic capabilities, including the ability to extract energy from otherwise indigestible dietary polysaccharides. Studies of a few unrelated, healthy adults have revealed substantial diversity in their gut communities, as measured by sequencing 16S rRNA genes, yet how this diversity relates to function and to the rest of the genes in the collective genomes of the microbiota (the gut microbiome) remains obscure. Studies of lean and obese mice suggest that the gut microbiota affects energy balance by influencing the efficiency of calorie harvest from the diet, and how this harvested energy is used and stored. Here we characterize the faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity, and their mothers, to address how host genotype, environmental exposure and host adiposity influence the gut microbiome. Analysis of 154 individuals yielded 9,920 near full-length and 1,937,461 partial bacterial 16S rRNA sequences, plus 2.14 gigabases from their microbiomes. The results reveal that the human gut microbiome is shared among family members, but that each person's gut microbial community varies in the specific bacterial lineages present, with a comparable degree of co-variation between adult monozygotic and dizygotic twin pairs. However, there was a wide array of shared microbial genes among sampled individuals, comprising an extensive, identifiable 'core microbiome' at the gene, rather than at the organismal lineage, level. Obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity and altered representation of bacterial genes and metabolic pathways. These results demonstrate that a diversity of organismal assemblages can nonetheless yield a core microbiome at a functional level, and that deviations from this core are associated with different physiological states (obese compared with lean). ©2009 Macmillan Publishers Limited. All rights reserved.

Pyrosequencing of cDNA from brains of parrots with proventricular dilatation disease (PDD), an unexplained fatal inflammatory central, autonomic, and peripheral nervous system disease, showed 2 strains of a novel Borna virus. Real-time PCR confirmed virus presence in brain, proventriculus, and adrenal gland of 3 birds with PDD but not in 4 unaffected birds.

Structural variation of the genome involves kilobase- to megabase-sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements. We introduce high-throughput and massive paired-end mapping (PEM), a large-scale genome-sequencing method to identify structural variants (SVs) ∼3 kilobases (kb) or larger that combines the rescue and capture of paired ends of 3-kb fragments, massive 454 sequencing, and a computational approach to map DNA reads onto a reference genome. PEM was used to map SVs in an African and in a putatively European individual and identified shared and divergent SVs relative to the reference genome. Overall, we fine-mapped more than 1000 SVs and documented that the number of SVs among humans is much larger than initially hypothesized; many of the SVs potentially affect gene function. The breakpoint junction sequences of more than 200 SVs were determined with a novel pooling strategy and computational analysis. Our analysis provided insights into the mechanisms of SV formation in humans.

In colony collapse disorder (CCD), honey bee colonies inexplicably lose their workers. CCD has resulted in a loss of 50 to 90% of colonies in beekeeping operations across the United States. The observation that irradiated combs from affected colonies can be repopulated with naive bees suggests that infection may contribute to CCD. We used an unbiased metagenomic approach to survey microflora in CCD hives, normal hives, and imported royal jelly. Candidate pathogens were screened for significance of association with CCD by the examination of samples collected from several sites over a period of 3 years. One organism, Israeli acute paralysis virus of bees, was strongly correlated with CCD.

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

The complete sequencing of the human genome and the development of molecularly targeted cancer therapy have promoted efforts to identify systematically the genetic alterations in human cancer. By high-throughput sequencing of tyrosine kinase genes in human non-small-cell lung cancer, we identified somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene (EGFR) that are correlated with clinical response to EGFR tyrosine kinase inhibitors (TKIs). We have shown that these mutant forms of EGFR induce oncogenic transformation in different cellular systems. Cells whose growth depends on EGFR with mutations in exons 19 and 21 are sensitive to EGFR-TKIs, whereas cells expressing insertion mutations in exon 20 or the T790M point mutant, found in tumor biopsies from patients that relapsed after an initial response to EGFR-TKIs, are resistant. Furthermore, by applying a novel, massively parallel sequencing technology, we have shown that clinically relevant oncogene mutations can be detected in clinical specimens with very low tumor content, thereby enabling optimal patient selection for mutation-directed therapy. In summary, by applying high-throughput genomic resequencing, we have identified a novel therapeutic target, mutant EGFR, in lung cancer and evaluated its role in predicting response to targeted therapy.

Whole genome amplification methods are used to generate the large amounts of DNA that are required for genetic testing. Preparation of genomic DNA from clinical samples is a bottleneck in high-throughput genotyping and is frequently limited by the amount of specimen available. Precious DNA collections used for association and linkage analysis can be a nonrenewable resource available to only a limited number of laboratories. To address these needs, amplified DNA is now being used in a growing number of research and diagnostic applications. A new method, called multiple displacement amplification, dramatically improves the high-fidelity reproduction of genomic DNA, with 10–100 kb amplified DNA products providing uniform coverage of genes.

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

The genetic relatedness of mountain gorillas and humans has led to concerns about interspecies transmission of infectious agents. Human-to-gorilla transmission may explain human metapneumovirus in 2 wild mountain gorillas that died during a respiratory disease outbreak in Rwanda in 2009. Surveillance is needed to ensure survival of these critically endangered animals.

The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge.

We have extended our earlier work to show that individual 14-20mer peptide nucleic acid probes directed against interspersed alpha-satellite sequences can specifically identify chromosomes. Peptide nucleic acid (PNA) probes were used to detect chromosomal abnormalities and repeat structure in the human genome by fluorescence in situ hybridization (FISH). The hybridization of a single PNA probe species directed against a highly abundant alpha-satellite DNA repeat sequence was sufficient to absolutely identify a chromosome. Selection of highly repetitive or region-specific DNA repeats involved DNA database analysis. Distribution of a specific repeat sequence in human genome was estimated through two means: a computer program "whole genome" approach based on approximately 400 Mb (12%) human genomic sequence. The other method involved directed search for alpha satellite sequences. In total, approximately 240 unique DNA repeat candidates were found. Forty-two PNA probes were designed for screening chromosome-specific probes. Ten chromosome-specific PNA probes for human Chromosomes (Chrs) 1, 2, 7, 9, 11, 17, 18, X, and Y have been identified. Interphase and metaphase results demonstrate that chromosome-specific PNA probes are capable of detecting simple aneuploidies (trisomies) in human. Another set of PNA probes showed distinct banding-like patterns and could be used as sequence-specific stains for chromosome "bar coding". Potential application of PNA probes for investigating repeat structure and function is also discussed.

The satellite repeat structure of the mammalian centromere contains the CENP-B protein binding site. Using the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), we show by direct PNA-DNA binding that all detectable CENP-B sites in a mammalian genome might have the same sequence. Two species-specific PNA 17-mers, pMm and pMc, were identified from CENP-B binding sites of Mus musculus and M. caroli, respectively. Fluorescence in situ hybridization confirmed that pMc hybridized to M. caroli centromeres only; however, pMm cross-hybridized to M. musculus and human centromeres. By using a series of CENP-B PNA 17-mers containing 1, 2, 3, 5, and 7 base-pair mismatches to their DNA counterparts, we further demonstrate that PNA-FISH can discriminate between two CENP-B DNA sequences that differ by a single base-pair in mouse and human centromeres, suggesting the degree of conservation of CENP-B sequences throughout the genome. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibility, and lower background. Therefore, PNA oligomers have significant advantages over DNA oligonucleotide probes in analyzing microsatellites in a genome.

Peptide nucleic acids (PNAs) can bind to single-stranded DNA by Watson–Crick base pairing and can form triple helices via Hoogsteen bonding to DNA/PNA duplexes. A single dimeric PNA molecule can form a clamp via both double- and triple-helix formation. We designed PNAs to bind as clamps to a site in the supFG1 mutation reporter gene carried within a chromosomally integrated, recoverable λ phage shuttle vector in mouse fibroblasts. The PNAs were introduced into the cells via permeabilization with streptolysin-O, and cellular uptake was confirmed by fluorescein labeling and fluorescent microscopy. PNAs specific for either an 8- or a 10-bp site in the supFG1 gene were found to induce mutations at frequencies in the range of 0.1%, 10-fold above the background. PNAs with three or four mismatches showed poor in vitro target site binding and were ineffective in the mutagenesis assay. No increased mutagenesis was detected with any of the PNAs in the nontargeted cII gene, also carried within the λ vector, further indicating the specificity of the PNA-induced mutagenesis. DNA sequence analysis revealed that the majority of the mutations were located within the PNA-binding site and consisted mostly of single base pair insertions and deletions within the poly G:C run there, suggesting that a high affinity PNA clamp constitutes a mutagenic lesion that may provoke replication slippage errors. The ability to direct mutations to a target site in chromosomal DNA by using PNAs may provide a useful tool for research and therapeutic applications.

Three protocols in this issue highlight applications of emulsification procedures, which deliver high-throughput potential to the molecular biology laboratory, without the need for automation. These procedures have already generated interesting results and spurred the development of exciting new technologies, while requiring only readily available laboratory equipment.

The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide with the linker in conventional phosphoamidite or H-phosphonate DNA syntheses. Directly, or via a post modification step, an oligonucleotide is labelled with one or more reporter moieties, e.g. dansyl (5-dimethylamino)-1-naphthalenesulfonyl), biotin, digoxigenin, DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-1-diazole (NBD), or pyrene. The present invention also relates to a solid phase support, e.g. a Controlled Pore Glass (CPG), immobilized linker reagent, to a method for preparing a labelled oligonucleotide, and to the use of the labelled oligonucleotide as hybridisation probe, in polymerase chain reactions (PCR), in nucleic acid sequencing, in cloning recombinant DNA and in vitro mutagenesis.

The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Endophytes represent a promising group of organisms, as they are a mostly untapped reservoir of metabolic diversity. They are often able to degrade cellulose, and they can produce an extraordinary diversity of metabolites. The filamentous fungal endophyte Ascocoryne sarcoides was shown to produce potential-biofuel metabolites when grown on a cellulose-based medium; however, the genetic pathways needed for this production are unknown and the lack of genetic tools makes traditional reverse genetics difficult. We present the genomic characterization of A. sarcoides and use transcriptomic and metabolomic data to describe the genes involved in cellulose degradation and to provide hypotheses for the biofuel production pathways. In total, almost 80 biosynthetic clusters were identified, including several previously found only in plants. Additionally, many transcriptionally active regions outside of genes showed condition-specific expression, offering more evidence for the role of long non-coding RNA in gene regulation. This is one of the highest quality fungal genomes and, to our knowledge, the only thoroughly annotated and transcriptionally profiled fungal endophyte genome currently available. The analyses and datasets contribute to the study of cellulose degradation and biofuel production and provide the genomic foundation for the study of a model endophyte system., Author Summary A renewable source of energy is a pressing global need. The biological conversion of lignocellulose to biofuels by microorganisms presents a promising avenue, but few organisms have been studied thoroughly enough to develop the genetic tools necessary for rigorous experimentation. The filamentous-fungal endophyte A. sarcoides produces metabolites when grown on a cellulose-based medium that include eight-carbon volatile organic compounds, which are potential biofuel targets. Here we use broadly applicable methods including genomics, transcriptomics, and metabolomics to explore the biofuel production of A. sarcoides. These data were used to assemble the genome into 16 scaffolds, to thoroughly annotate the cellulose-degradation machinery, and to make predictions for the production pathway for the eight-carbon volatiles. Extremely high expression of the gene swollenin when grown on cellulose highlights the importance of accessory proteins in addition to the enzymes that catalyze the breakdown of the polymers. Correlation of the production of the eight-carbon biofuel-like metabolites with the expression of lipoxygenase pathway genes suggests the catabolism of linoleic acid as the mechanism of eight-carbon compound production. This is the first fungal genome to be sequenced in the family Helotiaceae, and A. sarcoides was isolated as an endophyte, making this work also potentially useful in fungal systematics and the study of plant–fungus relationships.

The effects of PNA (peptide nucleic acid) bound to double-stranded (ds) DNA targets positioned downstream from phage T3 or T7 promoters in pBluescriptKS+ derived plasmids on transcription by RNA polymerases T3 or T7 have been studied. The dsDNA targets A10, 5'-A5GA4 or 5'-A2GA2GA4, and the corresponding PNAs T10, T5CT4 and T2CT2CT4 were used and the target-PNA strand displacement complexes were performed in low-salt buffer, since PNA does not bind efficiently to ds DNA in higher salt than 50 mM. It is shown that transcription elongation is arrested at the target site with PNA bound to the template strand, whereas only a marginal effect is observed with PNA bound to the non-template strand. With PNA T10, transcription arrest occurs at the first base of the PNA-binding site, while the arrest with the PNA T5CT4 takes place 2-3 nt inside the PNA binding site. In the case of PNA T2CT2CT4 the arrest is less efficient and occurs at the last 1-3 nt of the binding site. Transcription arrest was also shown for PNAs T6 and T8, although with a much lower efficiency. These results show that efficient transcription elongation arrest can be obtained by PNA targeting of the template DNA strand.

The stability of a new type of DNA mimic, peptide nucleic acid (PNA) in human blood serum, Eschericia coli and Micrococcus luteus extracts and nuclear and cytoplasmic extracts from mouse Ehrlich ascites tumor cells was investigated using HPLC analysis. Under conditions that caused complete cleavage of a control peptide, adrenocorticotropic hormone fragment 4-10, no significant degradation of the PNAs, H-T10-LysNH2 and H-TGTACGTCACAACTA-NH2, could be detected. Similarly, PNA H-T5-LysNH2 was found to resist attack by fungal proteinase K or porcine intestinal mucosa peptidase at concentrations exceeding those necessary to completely degrade a control peptide, H-Phe-Trp-Tyr-Cys-Phe-Trp-Tyr-Lys-Phe-Trp-Tyr-Lys-OH, by at least 1000- and 30-fold, respectively. Thus PNA is expected to have sufficient biostability to be used as a drug.

The vast majority of wine fermentations are performed principally by Saccharomyces cerevisiae. However, there are a growing number of instances in which other species of Saccharomyces play a predominant role. Interestingly, the presence of these other yeast species generally occurs via the formation of interspecific hybrids that contain genomic contributions from both S. cerevisiae and non-S. cerevisiae species. However, despite the large number of wine strains that are characterized at the genomic level, there remains limited information regarding the detailed genomic structure of hybrids used in winemaking. To address this, we describe the genome sequence of the thiol-releasing commercial wine yeast hybrid VIN7. VIN7 is shown to be an almost complete allotriploid interspecific hybrid that is comprised of a heterozygous diploid complement of S. cerevisiae chromosomes and a haploid Saccharomyces kudriavzevii genomic contribution. Both parental strains appear to be of European origin, with the S. cerevisiae parent being closely related to, but distinct from, the commercial wine yeasts QA23 and EC1118. In addition, several instances of chromosomal rearrangement between S. cerevisiae and S. kudriavzevii sequences were observed that may mark the early stages of hybrid genome consolidation. Refereed/Peer-reviewed

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species., Author Summary The yeast S. cerevisiae has been associated with human activity for thousands of years in industries such as baking, brewing, and winemaking. During this time, humans have effectively domesticated this microorganism, with different industries selecting for specific desirable phenotypic traits. This has resulted in the species S. cerevisiae comprising a genetically diverse collection of individual strains that are often suited to very specific roles (e.g. wine strains produce wine but not beer and vice versa). In order to understand the genetic differences that underpin these diverse industrial characteristics, we have sequenced the genomes of six industrial strains of S. cerevisiae that comprise four strains used in commercial wine production and two strains used in beer brewing. By comparing these genome sequences to existing S. cerevisiae genome sequences from laboratory, pathogenic, bioethanol, and “natural” isolates, we were able to identify numerous genetic differences among these strains including the presence of novel open reading frames and genomic rearrangements, which may provide the basis for the phenotypic differences observed among these strains.

Bats are reservoirs for emerging zoonotic viruses that can have a profound impact on human and animal health, including lyssaviruses, filoviruses, paramyxoviruses, and severe acute respiratory syndrome coronaviruses (SARS-CoVs). In the course of a project focused on pathogen discovery in contexts where human-bat contact might facilitate more efficient interspecies transmission of viruses, we surveyed gastrointestinal tissue obtained from bats collected in caves in Nigeria that are frequented by humans. Coronavirus consensus PCR and unbiased high-throughput pyrosequencing revealed the presence of coronavirus sequences related to those of SARS-CoV in a Commerson’s leaf-nosed bat (Hipposideros commersoni). Additional genomic sequencing indicated that this virus, unlike subgroup 2b CoVs, which includes SARS-CoV, is unique, comprising three overlapping open reading frames between the M and N genes and two conserved stem-loop II motifs. Phylogenetic analyses in conjunction with these features suggest that this virus represents a new subgroup within group 2 CoVs., IMPORTANCE Bats (order Chiroptera, suborders Megachiroptera and Microchiroptera) are reservoirs for a wide range of viruses that cause diseases in humans and livestock, including the severe acute respiratory syndrome coronavirus (SARS-CoV), responsible for the global SARS outbreak in 2003. The diversity of viruses harbored by bats is only just beginning to be understood because of expanded wildlife surveillance and the development and application of new tools for pathogen discovery. This paper describes a new coronavirus, one with a distinctive genomic organization that may provide insights into coronavirus evolution and biology.

We deeply sampled the organismal, genetic, and transcriptional diversity in fecal samples collected from a monozygotic (MZ) twin pair and compared the results to 1,095 communities from the gut and other body habitats of related and unrelated individuals. Using a new scheme for noise reduction in pyrosequencing data, we estimated the total diversity of species-level bacterial phylotypes in the 1.2-1.5 million bacterial 16S rRNA reads obtained from each deeply sampled cotwin to be ~800 (35.9%, 49.1% detected in both). A combined 1.1 million read 16S rRNA dataset representing 281 shallowly sequenced fecal samples from 54 twin pairs and their mothers contained an estimated 4,018 species-level phylotypes, with each sample having a unique species assemblage (53.4 ± 0.6% and 50.3 ± 0.5% overlap with the deeply sampled cotwins). Of the 134 phylotypes with a relative abundance of >0.1% in the combined dataset, only 37 appeared in >50% of the samples, with one phylotype in the Lachnospiraceae family present in 99%. Nongut communities had significantly reduced overlap with the deeply sequenced twins’ fecal microbiota (18.3 ± 0.3%, 15.3 ± 0.3%). The MZ cotwins’ fecal DNA was deeply sequenced (3.8-6.3 Gbp/sample) and assembled reads were assigned to 25 genus-level phylogenetic bins. Only 17% of the genes in these bins were shared between the cotwins. Bins exhibited differences in their degree of sequence variation, gene content including the repertoire of carbohydrate active enzymes present within and between twins (e.g., predicted cellulases, dockerins), and transcriptional activities. These results provide an expanded perspective about features that make each of us unique life forms and directions for future characterization of our gut ecosystems.

To examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of hESCs into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation—N1 (early initiation), N2 (neural progenitor), and N3 (early glial-like)—were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call isoform specialization . During neural differentiation, we observed differential expression of many types of genes, including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, neural progenitor cell identity maintenance, and the transition from a predominantly neuronal state into one with increased gliogenic potential.

International audience; We report a high-quality draft genome sequence of the domesticated apple (Malus x domestica). We show that a relatively recent (> 50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

Background It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (

Genome-wide association studies (GWASs) are regularly used to map genomic regions contributing to common human diseases, but they often do not identify the precise causative genes and sequence variants. To identify causative type 1 diabetes (T1D) variants, we resequenced exons and splice sites of 10 candidate genes in pools of DNA from 480 patients and 480 controls and tested their disease association in over 30,000 participants. We discovered four rare variants that lowered T1D risk independently of each other (odds ratio = 0.51 to 0.74; P = 1.3 × 10 –3 to 2.1 × 10 –16 ) in IFIH1 ( interferon induced with helicase C domain 1 ), a gene located in a region previously associated with T1D by GWASs. These variants are predicted to alter the expression and structure of IFIH1 [MDA5 (melanoma differentiation-associated protein 5)], a cytoplasmic helicase that mediates induction of interferon response to viral RNA. This finding firmly establishes the role of IFIH1 in T1D and demonstrates that resequencing studies can pinpoint disease-causing genes in genomic regions initially identified by GWASs.

Recent steep declines in honey bee health have severely impacted the beekeeping industry, presenting new risks for agricultural commodities that depend on insect pollination. Honey bee declines could reflect increased pressures from parasites and pathogens. The incidence of the microsporidian pathogen Nosema ceranae has increased significantly in the past decade. Here we present a draft assembly (7.86 MB) of the N. ceranae genome derived from pyrosequence data, including initial gene models and genomic comparisons with other members of this highly derived fungal lineage. N. ceranae has a strongly AT-biased genome (74% A+T) and a diversity of repetitive elements, complicating the assembly. Of 2,614 predicted protein-coding sequences, we conservatively estimate that 1,366 have homologs in the microsporidian Encephalitozoon cuniculi, the most closely related published genome sequence. We identify genes conserved among microsporidia that lack clear homology outside this group, which are of special interest as potential virulence factors in this group of obligate parasites. A substantial fraction of the diminutive N. ceranae proteome consists of novel and transposable-element proteins. For a majority of well-supported gene models, a conserved sense-strand motif can be found within 15 bases upstream of the start codon; a previously uncharacterized version of this motif is also present in E. cuniculi. These comparisons provide insight into the architecture, regulation, and evolution of microsporidian genomes, and will drive investigations into honey bee–Nosema interactions., Author Summary Honey bee colonies are in decline in many parts of the world, in part due to pressures from a diverse assemblage of parasites and pathogens. The range and prevalence of the microsporidian pathogen Nosema ceranae has increased significantly in the past decade. Here we describe the N. ceranae genome, presenting genome traits, gene models and regulatory motifs. N. ceranae has an extremely reduced and AT-biased genome, yet one with substantial numbers of repetitive elements. We identify novel genes that appear to be conserved among microsporidia but undetected outside this phylum, which are of special interest as potential virulence factors for these obligate pathogens. A previously unrecognized motif is found upstream of many start codons and likely plays a role in gene regulation across the microsporidia. These and other comparisons provide insight into the architecture, regulation, and evolution of microsporidian genomes, and provide the first genetic tools for understanding how this pathogen interacts with honey bee hosts.

A series of reagents containing 3- or 4-nitrobenzamido ligands tethered to 9-aminoacridine via variable-length linkers have been prepared and their properties as photochemical DNA cleavers (photonucleases) examined. When irradiated with approximately 300-nm light, where the nitrobenzamido ligand can absorb, they cleave DNA in an oxygen-independent reaction presumably involving oxygen transfer from the nitro group to the deoxyribose units of the DNA backbone (Nielsen et al., 1988b). This reaction is pH independent and only slightly affected by the linker length, and the DNA fragments are not substrates for DNA polymerase. When approximately 420-nm light is used, were only the 9-aminoacridinyl ligands absorb, the DNA cleavage is also oxygen-independent but pH dependent, requires DNA saturation with the reagent (base pair:reagent less than or equal to 2), and is most efficient with the longer linkers. The cleavage is specific for guanine residues and results in 5'-phosphate termini and heterogeneous (more than four products) 3'-termini. One of the products is presumably 3'-hydroxy since DNA photocleaved with nitrobenzamido acridine reagents and 420-nm radiation are substrates for DNA polymerase in a nick translation assay as well as for the Klenow fragment. An electron-transfer mechanism is suggested.

Three patients who received visceral-organ transplants from a single donor on the same day died of a febrile illness 4 to 6 weeks after transplantation. Culture, polymerase-chain-reaction (PCR) and serologic assays, and oligonucleotide microarray analysis for a wide range of infectious agents were not informative.We evaluated RNA obtained from the liver and kidney transplant recipients. Unbiased high-throughput sequencing was used to identify microbial sequences not found by means of other methods. The specificity of sequences for a new candidate pathogen was confirmed by means of culture and by means of PCR, immunohistochemical, and serologic analyses.High-throughput sequencing yielded 103,632 sequences, of which 14 represented an Old World arenavirus. Additional sequence analysis showed that this new arenavirus was related to lymphocytic choriomeningitis viruses. Specific PCR assays based on a unique sequence confirmed the presence of the virus in the kidneys, liver, blood, and cerebrospinal fluid of the recipients. Immunohistochemical analysis revealed arenavirus antigen in the liver and kidney transplants in the recipients. IgM and IgG antiviral antibodies were detected in the serum of the donor. Seroconversion was evident in serum specimens obtained from one recipient at two time points.Unbiased high-throughput sequencing is a powerful tool for the discovery of pathogens. The use of this method during an outbreak of disease facilitated the identification of a new arenavirus transmitted through solid-organ transplantation.

Next-generation sequencing technologies are revolutionizing human genomics, promising to yield draft genomes cheaply and quickly. One such technology has now been used to analyse much of the genetic code of a single individual — who happens to be James D. Watson. The procedure, which involves no cloning of the genomic DNA, makes use of the latest 454 parallel sequencing instrument. The sequence cost less than US$1 million (and a mere two months) to produce, compared to the approximately US$100 million reported for sequencing Craig Venter's genome by traditional methods. Still a major undertaking, but another step towards the goal of 'personalized genomes' and 'personalized medicine'. The DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels is reported. The association of genetic variation with disease and drug response, and improvements in nucleic acid technologies, have given great optimism for the impact of ‘genomic medicine’. However, the formidable size of the diploid human genome1, approximately 6 gigabases, has prevented the routine application of sequencing methods to deciphering complete individual human genomes. To realize the full potential of genomics for human health, this limitation must be overcome. Here we report the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. This sequence was completed in two months at approximately one-hundredth of the cost of traditional capillary electrophoresis methods. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence. In addition, we accurately identified small-scale (2–40,000 base pair (bp)) insertion and deletion polymorphism as well as copy number variation resulting in the large-scale gain and loss of chromosomal segments ranging from 26,000 to 1.5 million base pairs. Overall, these results agree well with recent results of sequencing of a single individual2 by traditional methods. However, in addition to being faster and significantly less expensive, this sequencing technology avoids the arbitrary loss of genomic sequences inherent in random shotgun sequencing by bacterial cloning because it amplifies DNA in a cell-free system. As a result, we further demonstrate the acquisition of novel human sequence, including novel genes not previously identified by traditional genomic sequencing. This is the first genome sequenced by next-generation technologies. Therefore it is a pilot for the future challenges of ‘personalized genome sequencing’.

The presence of workers that forgo reproduction and care for their siblings is a defining feature of eusociality and a major challenge for evolutionary theory. It has been proposed that worker behavior evolved from maternal care behavior. We explored this idea by studying gene expression in the primitively eusocial wasp Polistes metricus. Because little genomic information existed for this species, we used 454 sequencing to generate 391,157 brain complementary DNA reads, resulting in robust hits to 3017 genes from the honey bee genome, from which we identified and assayed orthologs of 32 honey bee behaviorally related genes. Wasp brain gene expression in workers was more similar to that in foundresses, which show maternal care, than to that in queens and gynes, which do not. Insulin-related genes were among the differentially regulated genes, suggesting that the evolution of eusociality involved major nutritional and reproductive pathways.

The limited life span of most blood cells requires the continuous production of cells, which in adults exceeds 10(12) cells/day. This impressive production of cells (approximately 4 x 10(16) cells over a lifetime) is achieved by the proliferation and differentiation of committed progenitor cells, which themselves are derived from a population of pluripotent stem cells with self-renewal potential. Paradoxically, the large majority of stem cells in adult bone marrow are quiescent cells. One possibility is that stem cells, like other somatic cells, have only a limited replicative potential (< 100 divisions). This hypothesis is supported by two key observations and the consideration that, in theory, 55 divisions can yield 4 x 10(16) cells. First, it was shown that 'candidate' stem cells purified from fetal and adult tissue showed dramatic functional differences in turn-over time and the ability to produce cells with stem cell properties, Second, these functional differences were found to correlate with a measurable loss of telomere repeats despite the presence of low but readily detectable levels of telomerase in all purified cell fractions. In order to address questions about the role of telomeres in normal and malignant haemopoiesis, we developed a quantitative fluorescence in situ hybridization technique. Here we review the characteristics of this novel tool to assess the number of telomere repeats at the end of individual chromosomes and provide an overview of recent observations.

BackgroundWhole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes.Halobacteriumspecies NRC-1 DNA andCampylobacter jejuniwere amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls.ResultsAll amplification methodologies induced statistically significant bias relative to the unamplified control. For theHalobacteriumspecies NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. ForCampylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR.ConclusionOf the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.

Background Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. Results A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point. Conclusions Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

Preparation of genomic DNA from clinical samples is a bottleneck in genotyping and DNA sequencing analysis and is frequently limited by the amount of specimen available. We use Multiple Displacement Amplification (MDA) to amplify the whole genome 10,000-fold directly from small amounts of whole blood, dried blood, buccal cells, cultured cells, and buffy coats specimens, generating large amounts of DNA for genetic testing. Genomic DNA was evenly amplified with complete coverage and consistent representation of all genes. All 47 loci analyzed from 44 individuals were represented in the amplified DNA at between 0.5- and 3.0-fold of the copy number in the starting genomic DNA template. A high-fidelity DNA polymerase ensures accurate representation of the DNA sequence. The amplified DNA was indistinguishable from the original genomic DNA template in 5 SNP and 10 microsatellite DNA assays on three different clinical sample types for 20 individuals. Amplification of genomic DNA directly from cells is highly reproducible, eliminates the need for DNA template purification, and allows genetic testing from small clinical samples. The low amplification bias of MDA represents a dramatic technical improvement in the ability to amplify a whole genome compared with older, PCR-based methods.

Site-specific DNA binding molecules offer the potential for genetic manipulation of mammalian cells. Peptide nucleic acids (PNAs) are a DNA mimic in which the purine and pyrimidine bases are attached to a polyamide backbone. PNAs bind with high affinity to single-stranded DNA via Watson–Crick base pairing and can form triple helices via Hoogsteen binding to DNA/PNA duplexes. Dimeric bis-PNAs capable of both strand invasion and triplex formation can form clamp structures on target DNAs. As a strategy to promote site-directed recombination, a bis-PNA was coupled to a 40-nt donor DNA fragment homologous to an adjacent region in the target gene. The PNA–DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency at least 60-fold above background. Induced site-specific recombination was also seen when the bis-PNA and the donor DNA were co-mixed without covalent linkage. In addition, the bis-PNA and the bis-PNA–DNA conjugate were found to induce DNA repair specifically in the target plasmid. Both the PNA-induced recombination and the PNA-induced repair were found to be dependent on the nucleotide excision repair factor, XPA (xeroderma pigmentosum complementation group A protein). These results suggest that the formation of a PNA clamp on duplex DNA creates a helical distortion that strongly provokes DNA repair and thereby sensitizes the target site to recombination. The ability to promote recombination in a site-directed manner using PNA–DNA conjugates may provide a useful strategy to achieve targeted correction of defective genes.

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO 4 , 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO 4 , 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ≥100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10 −21 M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.

Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and biosensors.