Torstein Tengs

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing

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. NOTE: In the version of this article initially published, it should have been acknowledged that Jan F. Simons, in addition to Roman K. Thomas and Elizabeth Nickerson, contributed equally to this work. The error has been corrected in the HTML and PDF versions of the article.

Development of Real-Time PCR Assays for Rapid Detection of Pfiesteria piscicida and Related Dinoflagellates

ABSTRACT Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far,Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteriaspecies, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugols solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.

Heart and skeletal muscle inflammation of farmed salmon is associated with infection with a novel reovirus

Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999[1], HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom[2]. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Kochs postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

Phenotypically different microalgal morphospecies with identical ribosomal DNA: a case of rapid adaptive evolution?

The agents driving the divergence and speciation of free-living microbial populations are still largely unknown. We investigated the dinoflagellate morphospecies Scrippsiella hangoei and Peridinium aciculiferum, which abound in the Baltic Sea and in northern temperate lakes, respectively. Electron microscopy analyses showed significant interspecific differences in the external cellular morphology, but a similar plate pattern in the characteristic dinoflagellate armor. Experimentally, S. hangoei grew in a wide range of salinities (0–30), whereas P. aciculiferum only grew in low salinities (0–3). Despite these phenotypic differences and the habitat segregation, molecular analyses showed identical ribosomal DNA sequences (ITS1, ITS2, 5.8S, SSU, and partial LSU) for both morphospecies. Yet, a strong interspecific genetic isolation was indicated by amplified fragment length polymorphism (FST = 0.76) and cytochrome b (cob) sequence divergence (∼1.90%). Phylogenetic reconstructions based on ribosomal (SSU, LSU) and mitochondrial (cob) DNA indicated a recent marine ancestor for P. aciculiferum. In conclusion, we suggest that the lacustrine P. aciculiferum and the marine-brackish S. hangoei diverged very recently, after a marine–freshwater transition that exposed the ancestral populations to different selective pressures. This hypothetical scenario agrees with mounting data indicating a significant role of natural selection in the divergence of free-living microbes, despite their virtually unrestricted dispersal capabilities. Finally, our results indicate that identical ITS rDNA sequences do not necessarily imply the same microbial species, as commonly assumed.

A quantitative TaqMan® MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci.

Here we present the development and first validation of a TaqMan minor groove binder (MGB) real-time polymerase chain reaction (RT-PCR) method for quantitative and highly specific detection of Aphanomyces astaci, the causative agent of crayfish plague. The assay specificity was experimentally assessed by testing against DNA representative of closely related oomycetes, and theoretically assessed by additional sequence similarity analyses comparing the primers and probe sequences to available sequences in EMBL/GenBank. The target of the assay is a 59 bp unique sequence motif of A. astaci found in the internal transcribed spacer 1 of the nuclear ribosomal gene cluster. A standard curve for quantification was established by setting up a four-fold dilution series with genomic A. astaci DNA. The absolute limit of detection (LOD(abs)), defined as the lowest concentration yielding a false negative probability<5% was found to be approximately 5 PCR forming units (PFU<or=target template copies) equivalent to less than one A. astaci genome. The absolute limit of quantification (LOQ(abs)) was experimentally established as 10 times the LOD(abs). Assay performance was also assessed with samples of naturally infected and non-infected susceptible crayfish (Astacus astacus) and carrier crayfish (Pacifastacus leniusculus). The benefits and limitations of the method are discussed, and guidance to practical application and interpretation of analytical results is provided.

A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS).

BackgroundCardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS.ResultsUsing high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. In situ hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus.ConclusionsAlthough causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS.

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real‐time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small‐subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.

SEQUESTRATION, PERFORMANCE, AND FUNCTIONAL CONTROL OF CRYPTOPHYTE PLASTIDS IN THE CILIATE MYRIONECTA RUBRA (CILIOPHORA)1

Myrionecta rubra (Lohmann 1908, Jankowski 1976 ) is a photosynthetic ciliate with a global distribution in neritic and estuarine habitats and has long been recognized to possess organelles of cryptophycean origin. Here we show, using nucleomorph (Nm) small subunit rRNA gene sequence data, quantitative PCR, and pigment absorption scans, that an M. rubra culture has plastids identical to those of its cryptophyte prey, Geminigera cf. cryophila (Taylor and Lee 1971, Hill 1991). Using quantitative PCR, we demonstrate that G. cf. cryophila plastids undergo division in growing M. rubra and are regulated by the ciliate. M. rubra maintained chl per cell and maximum cellular photosynthetic rates (Pmaxcell) that were 6–8 times that of G. cf. cryophila. While maximum chl‐specific photosynthetic rates (Pmaxchl) are identical between the two, M. rubra is less efficient at light harvesting in low light (LL) and has lower overall quantum efficiency. The photosynthetic saturation parameter (Ek) was not different between taxa in high light and was significantly higher in M. rubra in LL. Lower Chl:carbon ratios (θ), and hence PmaxC rates, in M. rubra resulted in lower growth rates compared with G. cf. cryophila. G. cf. cryophila possessed a greater capacity for synthesizing protein from photosynthate, while M. rubra used 3.2 times more fixed C for synthesizing lipids. Although cryptophyte plastids in M. rubra may not be permanently genetically integrated, they undergo replication and are regulated by M. rubra, allowing the ciliate to function as a phototroph.

Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production

The newly described piscine reovirus (PRV) appears to be associated with the development of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar L. PRV seems to be ubiquitous among fish in Norwegian salmon farms, but high viral loads and tissue distribution support a causal relationship between virus and disease. In order to improve understanding of the distribution of PRV in the salmon production line, we quantified PRV by using real-time PCR on heart samples collected at different points in the life cycle from pre-smolts to fish ready for slaughter. PRV positive pre-smolts were found in about 36% of the freshwater cohorts and a general increase in viral load was observed after their transfer to seawater. A reduction in viral loads was recorded when fish approached slaughter (18 mo in sea cages). Sequencing of positive samples did not support the hypothesis that outbreaks are caused by the spreading of a particular (virulent) strain of PRV.