Methane oxidation in industrial biogas plants-Insights in a novel methanotrophic environment evidenced by pmoA gene analyses and stable isotope labelling studies.

A broad methanotrophic community consisting of 16 different operational taxonomic units (OTUs) was detected by particulate methane monooxygenase A (pmoA) gene analyses of reactor sludge samples obtained from an industrial biogas plant. Using a cloning-sequencing approach, 75% of the OTUs were affiliated to the group of type I methanotrophs (γ-Proteobacteria) and 25% to type II methanotrophs (α-Proteobacteria) with a distinct predominance of the genus Methylobacter. By database matching, half of the total OTUs may constitute entirely novel species. For evaluation of process conditions that support growth of methanotrophic bacteria, qPCR analyses of pmoA gene copy numbers were performed during a sampling period of 70 days at varying reactor feeding scenarios. During the investigation period, methanotrophic cell counts estimated by qPCR fluctuated between 3.4 × 104 and 2 × 105 cells/mL with no distinct correlation to the organic loading rate, the amount of CH4, O2 and NH4-N. Methanotrophic activity was proofed even at low O2 levels (1%) by using stable carbon isotope labelling experiments of CH4 in batch experiments inoculated with reactor sludge. Supplementation of 13C labelled CH4 in the headspace of the reaction vials unambiguously confirmed the formation of 13C labelled CO2. Thus, industrial biogas reactors can be considered as a further methanotrophic habitat that exhibits a unique methanotrophic community which is specifically adapted to high CH4 and low O2 concentrations. To the best of our knowledge, our study is the first accurate detection and quantification of methanotrophic bacteria in industrial biogas reactors.

Genomics and prevalence of bacterial and archaeal isolates from biogas-producing microbiomes.

BACKGROUND: To elucidate biogas microbial communities and processes, the application of high-throughput DNA analysis approaches is becoming increasingly important. Unfortunately, generated data can only partialy be interpreted rudimentary since databases lack reference sequences. RESULTS: Novel cellulolytic, hydrolytic, and acidogenic/acetogenic Bacteria as well as methanogenic Archaea originating from different anaerobic digestion communities were analyzed on the genomic level to assess their role in biomass decomposition and biogas production. Some of the analyzed bacterial strains were recently described as new species and even genera, namely Herbinix hemicellulosilytica T3/55T, Herbinix luporum SD1DT, Clostridium bornimense M2/40T, Proteiniphilum saccharofermentans M3/6T, Fermentimonas caenicola ING2-E5BT, and Petrimonas mucosa ING2-E5AT. High-throughput genome sequencing of 22 anaerobic digestion isolates enabled functional genome interpretation, metabolic reconstruction, and prediction of microbial traits regarding their abilities to utilize complex bio-polymers and to perform specific fermentation pathways. To determine the prevalence of the isolates included in this study in different biogas systems, corresponding metagenome fragment mappings were done. Methanoculleus bourgensis was found to be abundant in three mesophilic biogas plants studied and slightly less abundant in a thermophilic biogas plant, whereas Defluviitoga tunisiensis was only prominent in the thermophilic system. Moreover, several of the analyzed species were clearly detectable in the mesophilic biogas plants, but appeared to be only moderately abundant. Among the species for which genome sequence information was publicly available prior to this study, only the species Amphibacillus xylanus, Clostridium clariflavum, and Lactobacillus acidophilus are of importance for the biogas microbiomes analyzed, but did not reach the level of abundance as determined for M. bourgensis and D. tunisiensis. CONCLUSIONS: Isolation of key anaerobic digestion microorganisms and their functional interpretation was achieved by application of elaborated cultivation techniques and subsequent genome analyses. New isolates and their genome information extend the repository covering anaerobic digestion community members.

Antioxidant capacity of phenolic compounds on human cell lines as affected by grape-tyrosinase and Botrytis-laccase oxidation.

Phenolic components (PCs) are well-known for their positive impact on human health. In addition to their action as radical scavengers, they act as activators for the intrinsic cellular antioxidant system. Polyphenol oxidases (PPOs) such as tyrosinase and laccase catalyze the enzymatic oxidation of PCs and thus, can alter their scavenging and antioxidative capacity. In this study, oxidation by tryosinase was shown to increase the antioxidant capacity of many PCs, especially those that lack adjacent aromatic hydroxyl groups. In contrast, oxidation by laccase tended to decrease the antioxidant capacity of red wine and distinct PCs. This was clearly demonstrated for p-coumaric acid and resveratrol, which is associated with many health benefits. While oxidation by tyrosinase increased their antioxidant activity laccase treatment resulted in a decreased activity and also of that for red wines.

Roles and Responsibilities in the Provision of Accredited Continuing Medical Education/Continuing Professional Development.

The Cologne Consensus Conference 2015 has focused on "Providers in accredited CME[continuing medical education]/CPD [continuing professional development]". As an outcome of the CCC 2015, the authors of this paper, who were part of the faculty, propose a contemporary definition of the roles and responsibilities of stakeholders involved in the different stages of planning, delivery and evaluation of CME/CPD.

Isolation of lactic acid-forming bacteria from biogas plants.

Direct molecular approaches provide hints that lactic acid bacteria play an important role in the degradation process of organic material to methanogenetic substrates in biogas plants. However, their diversity in biogas fermenter samples has not been analyzed in detail yet. For that reason, five different biogas fermenters, which were fed mainly with maize silage and manure from cattle or pigs, were examined for the occurrence of lactic acid-forming bacteria. A total of 197 lactic acid-forming bacterial strains were isolated, which we assigned to 21 species, belonging to the genera Bacillus, Clostridium, Lactobacillus, Pediococcus, Streptococcus and Pseudoramibacter-related. A qualitative multiplex system and a real-time quantitative PCR could be developed for most isolates, realized by the selection of specific primers. Their role in biogas plants was discussed on the basis of the quantitative results and on physiological data of the isolates.

Unraveling the microbiome of a thermophilic biogas plant by metagenome and metatranscriptome analysis complemented by characterization of bacterial and archaeal isolates.

BACKGROUND: One of the most promising technologies to sustainably produce energy and to mitigate greenhouse gas emissions from combustion of fossil energy carriers is the anaerobic digestion and biomethanation of organic raw material and waste towards biogas by highly diverse microbial consortia. In this context, the microbial systems ecology of thermophilic industrial-scale biogas plants is poorly understood. RESULTS: The microbial community structure of an exemplary thermophilic biogas plant was analyzed by a comprehensive approach comprising the analysis of the microbial metagenome and metatranscriptome complemented by the cultivation of hydrolytic and acido-/acetogenic Bacteria as well as methanogenic Archaea. Analysis of metagenome-derived 16S rRNA gene sequences revealed that the bacterial genera Defluviitoga (5.5 %), Halocella (3.5 %), Clostridium sensu stricto (1.9 %), Clostridium cluster III (1.5 %), and Tepidimicrobium (0.7 %) were most abundant. Among the Archaea, Methanoculleus (2.8 %) and Methanothermobacter (0.8 %) were predominant. As revealed by a metatranscriptomic 16S rRNA analysis, Defluviitoga (9.2 %), Clostridium cluster III (4.8 %), and Tepidanaerobacter (1.1 %) as well as Methanoculleus (5.7 %) mainly contributed to these sequence tags indicating their metabolic activity, whereas Hallocella (1.8 %), Tepidimicrobium (0.5 %), and Methanothermobacter (<0.1 %) were transcriptionally less active. By applying 11 different cultivation strategies, 52 taxonomically different microbial isolates representing the classes Clostridia, Bacilli, Thermotogae, Methanomicrobia and Methanobacteria were obtained. Genome analyses of isolates support the finding that, besides Clostridium thermocellum and Clostridium stercorarium, Defluviitoga tunisiensis participated in the hydrolysis of hemicellulose producing ethanol, acetate, and H2/CO2. The latter three metabolites are substrates for hydrogentrophic and acetoclastic archaeal methanogenesis. CONCLUSIONS: Obtained results showed that high abundance of microorganisms as deduced from metagenome analysis does not necessarily indicate high transcriptional or metabolic activity, and vice versa. Additionally, it appeared that the microbiome of the investigated thermophilic biogas plant comprised a huge number of up to now unknown and insufficiently characterized species.

Sugiyamaella mastotermitis sp. nov. and Papiliotrema odontotermitis f.a., sp. nov. from the gut of the termites Mastotermes darwiniensis and Odontotermes obesus.

Two novel yeast species were isolated from the guts of two different termite species. A new member of the genus Sugiyamaella was isolated from the hindgut and nest material of the lower Australian termite Mastotermes darwiniensis. The second novel yeast species, isolated from the higher termite Odontotermes obesus, was identified as a member of the genus Papiliotrema. Both yeast species were able to hydrolyse xylan, methylumbelliferyl ß-xylobiose and methylumbelliferyl ß-xylotriose. The ability to debranch different hemicellulose side chains and growth without the addition of external vitamins was observed. A symbiotic role of the novel yeast species is indicated, especially in respect to xylan degradation and the production of vitamins. Here, we describe these species as Sugiyamaella mastotermitis sp. nov., MycoBank 816574 (type strain MD39VT=DSM 100793T=CBS 14182T), and Papiliotrema odontotermitis f.a., sp. nov., MycoBank 816575 (type strain OO5T=DSM 100791T=CBS 14181T). Additionally, we transfer Candida qingdaonensis to the genus Sugiyamaella and propose the following combination: Sugiyamaella qingdaonensis f.a., comb. nov., MycoBank 816576.

Analysis of propionate-degrading consortia from agricultural biogas plants.

In order to investigate the propionate-degrading community of agricultural biogas plants, four propionate-degrading consortia (Ap1a, N12, G12, and Wp2a) were established from different biogas plants which were fed with renewable resources. The consortia were cultivated in a batch for a period of 2-4 years and then analyzed in an 8-week batch experiment for microbial succession during propionate degradation. Community shifts showed considerable propagation of Syntrophobacter sulfatireducens, Cryptanaerobacter sp./Pelotomaculum sp., and "Candidatus Cloacamonas sp." in the course of decreasing propionate concentration. Methanogenic species belonged mainly to the genera Methanosarcina, Methanosaeta, and Methanoculleus. Due to the prevalent presence of the syntrophic acetate-oxidizing species Tepidanaerobacter acetatoxydans and potentially autotrophic homoacetogenic bacteria (Moorella sp., Thermacetogenium sp.), a theoretical involvement of syntrophic acetate oxidation and autotrophic homoacetogenesis in stable and efficient propionate degradation was indicated. Considering theoretical Gibbs free energy values at different hydrogen partial pressures, it is noticeable that syntrophic acetate oxidation and autotrophic homoacetogenesis have the potential to counterbalance adverse hydrogen partial pressure fluctuations, stabilizing most probably continuous and stable propionate degradation.

Genomic characterization of Defluviitoga tunisiensis L3, a key hydrolytic bacterium in a thermophilic biogas plant and its abundance as determined by metagenome fragment recruitment.

The genome sequence of Defluviitoga tunisiensis L3 originating from a thermophilic biogas-production plant was established and recently published as Genome Announcement by our group. The circular chromosome of D. tunisiensis L3 has a size of 2,053,097bp and a mean GC content of 31.38%. To analyze the D. tunisiensis L3 genome sequence in more detail, a phylogenetic analysis of completely sequenced Thermotogae strains based on shared core genes was performed. It appeared that Petrotoga mobilis DSM 10674(T), originally isolated from a North Sea oil-production well, is the closest relative of D. tunisiensis L3. Comparative genome analyses of P. mobilis DSM 10674(T) and D. tunisiensis L3 showed moderate similarities regarding occurrence of orthologous genes. Both genomes share a common set of 1351 core genes. Reconstruction of metabolic pathways important for the biogas production process revealed that the D. tunisiensis L3 genome encodes a large set of genes predicted to facilitate utilization of a variety of complex polysaccharides including cellulose, chitin and xylan. Ethanol, acetate, hydrogen (H2) and carbon dioxide (CO2) were found as possible end-products of the fermentation process. The latter three metabolites are considered to represent substrates for methanogenic Archaea, the key organisms in the final step of the anaerobic digestion process. To determine the degree of relatedness between D. tunisiensis L3 and dominant biogas community members within the thermophilic biogas-production plant, metagenome sequences obtained from the corresponding microbial community were mapped onto the L3 genome sequence. This fragment recruitment revealed that the D. tunisiensis L3 genome is almost completely covered with metagenome sequences featuring high matching accuracy. This result indicates that strains highly related or even identical to the reference strain D. tunisiensis L3 play a dominant role within the community of the thermophilic biogas-production plant.

Isolation of acetic, propionic and butyric acid-forming bacteria from biogas plants.

In this study, acetic, propionic and butyric acid-forming bacteria were isolated from thermophilic and mesophilic biogas plants (BGP) located in Germany. The fermenters were fed with maize silage and cattle or swine manure. Furthermore, pressurized laboratory fermenters digesting maize silage were sampled. Enrichment cultures for the isolation of acid-forming bacteria were grown in minimal medium supplemented with one of the following carbon sources: Na(+)-dl-lactate, succinate, ethanol, glycerol, glucose or a mixture of amino acids. These substrates could be converted by the isolates to acetic, propionic or butyric acid. In total, 49 isolates were obtained, which belonged to the phyla Firmicutes, Tenericutes or Thermotogae. According to 16S rRNA gene sequences, most isolates were related to Clostridium sporosphaeroides, Defluviitoga tunisiensis and Dendrosporobacter quercicolus. Acetic, propionic or butyric acid were produced in cultures of isolates affiliated to Bacillus thermoamylovorans, Clostridium aminovalericum, Clostridium cochlearium/Clostridium tetani, C. sporosphaeroides, D. quercicolus, Proteiniborus ethanoligenes, Selenomonas bovis and Tepidanaerobacter sp. Isolates related to Thermoanaerobacterium thermosaccharolyticum produced acetic, butyric and lactic acid, and isolates related to D. tunisiensis formed acetic acid. Specific primer sets targeting 16S rRNA gene sequences were designed and used for real-time quantitative PCR (qPCR). The isolates were physiologically characterized and their role in BGP discussed.

Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation.

During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D-DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially available S. cerevisiae strain. All strains examined showed (i) different fermentative behaviour, (ii) stress resistance as well as (iii) susceptibility to stuck fermentation which was reflected in significant differences in protein expression levels. During our research we identified differentially expressed proteins in 155 gel spots which correspond to 70 different protein functions. Differences of expression between strains were observed mainly among proteins involved in stress response, proteins degradation pathways, cell redox homeostasis and amino acids biosynthesis. Interestingly, the newly discovered triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus strain which has the ability to naturally restart stuck fermentation showed a very strong induction of expression of two proteolytic enzymes: Pep4 and Prc1 that appear as numerous isoforms on the gel image and which may be the key to its unique properties. This study is an important step towards the better understanding of wine fermentations at a molecular level.

Isolation of methanotrophic bacteria from termite gut.

The guts of termites feature suitable conditions for methane oxidizing bacteria (MOB) with their permanent production of CH4 and constant supply of O2 via tracheae. In this study, we have isolated MOB from the gut contents of the termites Incisitermes marginipennis, Mastotermes darwiniensis, and Neotermes castaneus for the first time. The existence of MOB was indicated by detecting pmoA, the gene for the particulate methane monooxygenase, in the DNA of gut contents. Fluorescence in situ hybridization and quantitative real-time polymerase chain reaction supported those findings. The MOB cell titer was determined to be 10(2)-10(3) per gut. Analyses of the 16S rDNA from isolates indicated close similarity to the genus Methylocystis. After various physiological tests and fingerprinting methods, no exact match to a known species was obtained, indicating the isolation of new MOB species. However, MALDI-TOF MS analyses revealed a close relationship to Methylocystis bryophila and Methylocystis parvus.

Influence of Laccase and Tyrosinase on the Antioxidant Capacity of Selected Phenolic Compounds on Human Cell Lines.

Polyphenolic compounds affect the color, odor and taste of numerous food products of plant origin. In addition to the visual and gustatory properties, they serve as radical scavengers and have antioxidant effects. Polyphenols, especially resveratrol in red wine, have gained increasing scientific and public interest due to their presumptive beneficial impact on human health. Enzymatic oxidation of phenolic compounds takes place under the influence of polyphenol oxidases (PPO), including tyrosinase and laccase. Several studies have demonstrated the radical scavenger effect of plants, food products and individual polyphenols in vitro, but, apart from resveratrol, such impact has not been proved in physiological test systems. Furthermore, only a few data exist on the antioxidant capacities of the enzymatic oxidation products of phenolic compounds generated by PPO. We report here first results about the antioxidant effects of phenolic substances, before and after oxidation by fungal model tyrosinase and laccase. In general, the common chemical 2,2-diphenyl-1-picrylhydrazyl assay and the biological tests using two different types of cell cultures (monocytes and endothelial cells) delivered similar results. The phenols tested showed significant differences with respect to their antioxidant activity in all test systems. Their antioxidant capacities after enzymatic conversion decreased or increased depending on the individual PPO used.

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains of Saccharomyces cerevisiae and interspecies hybrids.

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast.

Complete genome sequence of the strain Defluviitoga tunisiensis L3, isolated from a thermophilic, production-scale biogas plant.

An anaerobic, thermophilic bacterium belonging to the phylum Thermotogae was isolated from a rural, thermophilic biogas plant (54°C) producing methane-rich biogas from maize silage, barley, cattle and pig manure. Here we report the first complete genome sequence of the Defluviitoga tunisiensis strain L3, an isolate from the family Thermotogaceae. The strain L3 encodes several genes predicted to be involved in utilization of a large diversity of complex carbohydrates including cellobiose and xylan for the production of acetate, hydrogen (H2) and carbon dioxide (CO2). The genome sequence of D. tunisiensis L3 provides the basis for biotechnological exploitation of genetic determinants playing an important role in thermophilic fermentation processes utilizing renewable primary products.

The Potential of the Yeast Debaryomyces hansenii H525 to Degrade Biogenic Amines in Food.

Twenty-six yeasts from different genera were investigated for their ability to metabolize biogenic amines. About half of the yeast strains produced one or more different biogenic amines, but some strains of Debaryomyces hansenii and Yarrowia lipolytica were also able to degrade such compounds. The most effective strain D. hanseniii H525 metabolized a broad spectrum of biogenic amines by growing and resting cells. Degradation of biogenic amines by this yeast isolate could be attributed to a peroxisomal amine oxidase activity. Strain H525 may be useful as a starter culture to reduce biogenic amines in fermented food.

Insights into the annotated genome sequence of Methanoculleus bourgensis MS2(T), related to dominant methanogens in biogas-producing plants.

The final step of the biogas production process, the methanogenesis, is frequently dominated by members of the genus Methanoculleus. In particular, the species Methanoculleus bourgensis was identified to play a role in different biogas reactor systems. The genome of the type strain M. bourgensis MS2(T), originally isolated from a sewage sludge digestor, was completely sequenced to analyze putative adaptive genome features conferring competitiveness within biogas reactor environments to the strain. Sequencing and assembly of the M. bourgensis MS2(T) genome yielded a chromosome with a size of 2,789,773 bp. Comparative analysis of M. bourgensis MS2(T) and Methanoculleus marisnigri JR1 revealed significant similarities. The absence of genes for a putative ammonium uptake system may indicate that M. bourgensis MS2(T) is adapted to environments rich in ammonium/ammonia. Specific genes featuring predicted functions in the context of osmolyte production were detected in the genome of M. bourgensis MS2(T). Mapping of metagenome sequences derived from a production-scale biogas plant revealed that M. bourgensis MS2(T) almost completely comprises the genetic information of dominant methanogens present in the biogas reactor analyzed. Hence, availability of the M. bourgensis MS2(T) genome sequence may be valuable regarding further research addressing the performance of Methanoculleus species in agricultural biogas plants.

Complete genome sequence of the methanogenic neotype strain Methanobacterium formicicum MF(T.).

The neotype strain Methanobacterium formicicum MF(T) (DSM1535), a hydrogenotrophic methanogenic Archaeon, was isolated from a domestic sewage sludge digestor in Urbana (IL, USA). Here, the complete genome sequence of the methanogen is reported. The genome is 2,478,074bp in size, featuring a GC content of 41.23%. M. formicicum MF(T) encodes several genes predicted to be involved in adaptation to abiotic stress such as high osmolarity. The strain MF(T) is of biotechnological importance since M. formicicum strains are often found in production-scale biogas plants and it is suggested as a starter culture for the anaerobic biomethanation process.

The yeast Wickerhamomyces anomalus AS1 secretes a multifunctional exo-ß-1,3-glucanase with implications for winemaking.

A multifunctional exo-ß-1,3-glucanase (WaExg2) was purified from the culture supernatant of the yeast Wickerhamomyces anomalus AS1. The enzyme was identified by mass spectroscopic analysis of tryptic peptide fragments and the encoding gene WaEXG2 was sequenced. The latter codes for a protein of 427 amino acids, beginning with a probable signal peptide (17 aa) for secretion. The mature protein has a molecular mass of 47 456 Da with a calculated pI of 4.84. The somewhat higher mass of the protein in SDS-PAGE might be due to bound carbohydrates. Presumptive disulphide bridges confer a high compactness to the molecule. This explains the apparent smaller molecular mass (35 kDa) of the native enzyme determined by electrophoresis, whereas the unfolded form is consistent with the theoretical mass. Enzymatic hydrolysis of selected glycosides and glycans by WaExg2 was proved by TLC analysis of cleavage products. Glucose was detected as the sole hydrolysis product from laminarin, underlining that the enzyme acts as an exoglucanase. In addition, the enzyme efficiently hydrolysed small ß-linked glycosides (arbutin, esculin, polydatin, salicin) and disaccharides (cellobiose, gentiobiose). WaExg2 was active under typical wine-related conditions, such as low pH (3.5-4.0), high sugar concentrations (up to 20% w/v), high ethanol concentrations (10-15% v/v), presence of sulphites (up to 2 mm) and various cations. Therefore, the characterized enzyme might have multiple uses in winemaking, to increase concentrations of sensory and bioactive compounds by splitting glycosylated precursors or to reduce viscosity by hydrolysis of glycan slimes.

Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing.

OBJECTIVES: Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in ∼5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs. MATERIALS AND METHODS: We developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5' and 3' ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analyzed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR. RESULTS: The H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5' and 3' ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3' ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples. CONCLUSION: Multiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing.