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The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Three microalgae-based treatment technologies were applied for removing nutrients in piggery wastewater and CO2 in biogas simultaneously. Chlorella vulgaris (C. vulgaris), Scenedesmus obliquus (S. obliquus), and Neochloris oleoabundans (N. oleoabundans) were selected for mono-cultivation or co-cultivation with fungi or activated sludge. The effects of initial inoculum concentration (low of 62.06 ± 6.23 mg/L, medium of 121 ± 9.34 mg/L, and high of 180 ± 12.78 mg/L) of microalgae/algal symbiont on the pollutant removal efficiency were evaluated. The results showed that cultivation of S. obliquus with activated sludge had a relatively high CO2 and nutrients removal efficiency at a medium concentration of initial inoculum. The highest removal efficiency of total organic carbon, total nitrogen, and total phosphorus in piggery wastewater was 87.29%, 87.26%, and 90.17% by co-cultivation of S. obliquus with activated sludge. The highest CO2 removal of 64.28% could be achieved under co-cultivation of S. obliquus and fungi at medium initial inoculum concentrations. This work will be helpful to promote the study of microalgae-based biogas upgrading and piggery wastewater purification. PRACTITIONER POINTS: Nutrient and CO2 were efficiently removed in moderate microalgal initial inoculum. Scenedesmus obliquus-activated sludge cultivation achieved the highest nutrient removal. Co-cultivation of S. obliquus and fungi showed the highest CO2 removal. Zinc was removed efficiently in high microalgal initial inoculum. Chlorella vulgaris/S. obliquus-activated sludge performed economically.

In this study, an anaerobically digested effluent from kitchen waste with high concentrations of chemical oxygen demand (COD) and ammonia nitrogen was treated using combined processes of anaerobic digestion (AD), complete nitritation (CN), and anaerobic ammonium oxidation (ANAMMOX). The COD and nitrogen removal efficiency of each treatment unit were investigated. The feasibility of using the final treatment effluent to dilute the original wastewater was also discussed. Findings showed that as a pretreatment step, AD resulted in the decline in biodegradability and increase in NH4+- N concentration. CN was successfully and stably achieved for 106 days with an average nitritation rate of 95% by maintaining the dissolved oxygen at 2-3 mg/L and hydraulic retention time of 24 hr under 30 ± 1°C. High NH4+- N and NO2-- N . removal efficiencies of over 88% and 96% were attained in the following ANAMMOX reactor. The reflux of ANAMMOX-treated effluent for the dilution of raw wastewater or an influent of CN and ANAMMOX ensured the stable operation of the combined system. PRACTITIONER POINTS: Anaerobic digestion effluent of kitchen waste had low COD/ NH4+-N ratio and poor biodegradability. Stable and efficient nitritation was realized by controlling DO, HRT and TEMP. High NH4+-N and NO2--N removal efficiency were obtained by ANAMMOX process. Average nitrogen removal rate of 0.94 kg N/m3 /day were obtained by ANAMMOX. Reflux dilution with the effluent guaranteed the system's successful operation.

To investigate the effects of different microalgae and culture methods on the purification of domestic wastewater and biogas, Chlorella vulgaris and Scenedesmus obliquus were selected. Three different culture methods (monoculture, microalgal-fungi cocultivation, and microalgal-activated sludge cocultivation) were used to remove nutrients from four different domestic wastewaters and remove CO2 from raw biogas in a photobioreactor. The results show that the effluent from the centrate of pretreated urban wastewater (WW4) achieved the highest nutrient and CO2 removal efficiency. Cocultivation of C. vulgaris and activated sludge achieved the highest COD, TP, and CO2 removal efficiencies of 79.27%, 81.25%, and 60.39% with WW4, respectively. Cocultivation of C. vulgaris and fungi achieved the highest TN removal efficiency of 78.46% with WW4. The contents of C, N, and P in the microalgal-activated sludge symbiont after treatment exceeded 50%, 8%, and 0.8%, respectively. Highly economically efficient energy consumption was achieved with WW4 for both C. vulgaris and S. obliquus. Microalgal-activated sludge cocultivation was identified as the optimal option for the simultaneous purification of wastewater and biogas based on its high pollution and CO2 removal efficiency. This provides a reference for the microalgal-based purification of actual domestic wastewater and raw biogas. PRACTITIONER POINTS: Nutrient and CO2 were efficiently removed by C. vulgaris with activated sludge. CO2 was removed up to 60.4% and was economically viable. Cocultivation of C. vulgaris and fungi could achieve the highest TN removal with WW4.

The target localisation in a multistatic passive radar system using bistatic range measurements from multiple transmitter–receiver pairs is considered. A novel closed-form solution employing multidimensional scaling (MDS) analysis technique is proposed, which is based on the optimisation of a cost function related to the scalar product matrix in the classical MDS framework. The proposed solution is robust to large measurement noise. Simulation results demonstrate the proposed solution achieves a significant performance improvement over existing spherical interpolation, spherical intersection and two-step weighted least squares methods, and attains the Cramer–Rao lower bound at a relatively higher measurement noise level before the threshold effect occurs.

BACKGROUND This study aims to simultaneously reduce biogas slurry nutrient and upgrade biogas via microalgal production. Marine microalgae were grown in a culture medium containing a mixture of seawater and biogas slurry with CO2 from synthetic biogas. The effects of different ratios of biogas slurry and various influent concentrations of CH4 in synthetic biogas on the growth of Nannochloropsis sp. were examined. Nutrient removal and biogas upgrading were also studied under different influent CH4 concentrations. RESULTS Nannochloropsis sp. showed optimal growth in 40% biogas slurry and significantly enhanced growth in synthetic biogas with 50% CO2. The optimal range of influent CH4 concentration for the removal of biogas slurry nutrients was 45% to 50%. Notably, the influent CH4 concentration of 60% (v/v) provided an optimal CO2 concentration for Nannochloropsis sp. cultivation. Advantageous CO2 removal and biogas upgrading were observed under this condition. This treatment increased CH4 concentration in synthetic biogas from 60% to 93.58% after 6 days cultivation. CONCLUSION Appropriate control of the biogas slurry or seawater and CH4 concentrations in the influent is crucial to simultaneously optimize nutrient removal and biogas upgrading. © 2015 Society of Chemical Industry

Primary testicular diffuse large B cell lymphoma (PTL) is an aggressive malignancy that occurs in the immune-privileged anatomical site of the testis. We have previously shown that structural genomic rearrangements involving the MHC class II transactivator CIITA and programmed death ligands (PDLs) 1 and 2 are frequent across multiple B cell lymphoma entities. Specifically in PTL, we found rearrangements in the PDL locus by fluorescence in situ hybridization (FISH). However, breakpoint anatomy and rearrangement partners were undetermined, while CIITA rearrangements had not been reported previously in PTL. Here, we performed bacterial artificial chromosome capture sequencing on three archival, formalin-fixed, paraffin-embedded tissue biopsies, interrogating 20 known rearrangement hotspots in B cell lymphomas. We report novel CIITA, FOXP1 and PDL rearrangements involving IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Moreover, we present immunohistochemistry data supporting the association between PDL rearrangements and increased protein expression. Finally, using FISH, we show that CIITA (8/82; 10%) and FOXP1 (5/74; 7%) rearrangements are recurrent in PTL. In summary, we describe rearrangement frequencies and novel rearrangement partners of the CIITA, FOXP1 and PDL loci at base-pair resolution in a rare, aggressive lymphoma. Our data suggest immune-checkpoint inhibitor therapy as a promising intervention for PTL patients harbouring PDL rearrangements.

High-grade serous carcinoma (HGSC) is the most common and fatal form of ovarian cancer. While most tumours are highly sensitive to cytoreductive surgery and platinum- and taxane-based chemotherapy, the majority of patients experience recurrence of treatment-resistant tumours. The clonal origin and mutational adaptations associated with recurrent disease are poorly understood. We performed whole exome sequencing on tumour cells harvested from ascites at three time points (primary, first recurrence, and second recurrence) for three HGSC patients receiving standard treatment. Somatic point mutations and small insertions and deletions were identified by comparison to constitutional DNA. The clonal structure and evolution of tumours were inferred from patterns of mutant allele frequencies. TP53 mutations were predominant in all patients at all time points, consistent with the known founder role of this gene. Tumours from all three patients also harboured mutations associated with cell cycle checkpoint function and Golgi vesicle trafficking. There was convergence of germline and somatic variants within the DNA repair, ECM, cell cycle control, and Golgi vesicle pathways. The vast majority of somatic variants found in recurrent tumours were present in primary tumours. Our findings highlight both known and novel pathways that are commonly mutated in HGSC. Moreover, they provide the first evidence at single nucleotide resolution that recurrent HGSC arises from multiple clones present in the primary tumour with negligible accumulation of new mutations during standard treatment.

Complex genome rearrangements are frequently observed in cancer but their impact on tumor molecular biology is largely unknown. Recent studies have identified a new phenomenon involving the simultaneous generation of tens to hundreds of genomic rearrangements, called chromothripsis. To understand the molecular consequences of these events, we sequenced the genomes and transcriptomes of two prostate tumors exhibiting evidence of chromothripsis. We identified several complex fusion transcripts, each containing sequence from three different genes, originating from different parts of the genome. One such poly-gene fusion transcript appeared to be expressed from a chain of small genomic fragments. Furthermore, we detected poly-gene fusion transcripts in the prostate cancer cell line LNCaP, suggesting they may represent a common phenomenon. Finally in one tumor with chromothripsis, we identified multiple mutations in the p53 signaling pathway, expanding on recent work associating aberrant DNA damage response mechanisms with chromothripsis. Overall, our data show that chromothripsis can manifest as massively rearranged transcriptomes. The implication that multigenic changes can give rise to poly-gene fusion transcripts is potentially of great significance to cancer genetics. V C 2012 Wiley Periodicals, Inc.

Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co-deletion to identify new molecular features at base-pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed seven others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non-synonymous somatic mutations in 20 CIC-mutant oligodendrogliomas, nine were in exon 5 within an annotated DNA-interacting domain and three were in exon 20 within an annotated protein-interacting domain. The remaining nine were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co-deletion, and IDH1/2 mutation (p < 0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild-type tumours, in a background of 1p/19q co-deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation, and 1p/19q co-deletion.

The western black cottonwood (Populus trichocarpa) was the first tree to have its genome fully sequenced and has emerged as the model species for the study of secondary growth and wood formation. It is also a good candidate species for the production of lignocellulosic biofuels. Here, we present and make available to the research community the results of the sequencing of the transcriptome of developing xylem in 20 accessions with high-throughput next generation sequencing technology. We found over 0.5 million putative single nucleotide polymorphisms (SNPs) in 26,595 genes that are expressed in developing secondary xylem. More than two-thirds of all SNPs were found in annotated exons, with 18% and 14% in regions of the genome annotated as introns and intergenic, respectively, where only 3% and 4% of sequence reads mapped. This suggests that the current annotation of the poplar genome is remarkably incomplete and that there are many transcripts and novel genes waiting to be annotated. We hope that this resource will stimulate further research in expression profiling, detection of alternative splicing and adaptive evolution in poplar.

To investigate the water circulation of eastern Qinghai-Tibet plateau during rainy season, water samples of precipitation, throughfall, fog, soil, litter and xylem were collected for stable isotope analysis. The results showed that precipitation mainly originated as a result of the East Asian Monsoon, and the secondarily evaporated water from subalpine ecosystem was an important part in local atmospheric water cycle. The deuterium excess of rainfall in the alpine meadow was evidently higher than the precipitation in the Dengsheng stations. This suggests that a large part of precipitation in alpine meadow was derived from secondarily evaporated water and the mean contribution was 39·57%, about 3·65 mm produced shortly after rain events. Through the contrast of delta (d)-excess value in different water samples, it could be concluded that the water in subalpine shrubland and transpiration of subalpine dark coniferous forest were the main source of secondarily evaporated water that transferred to alpine meadow. Hence, the precipitation on the east Qinghai-Tibet plateau was doubly controlled by monsoon and local water circulation in alpine ecosystems.

N-methyl-D-aspartate (NMDA) receptors play a key role in excitatory neurotransmission and are linked to a variety of acute and chronic neurodegenerative diseases including epilepsy, schizophrenia, Parkinson's disease and drug abuse. N-(2-chloro-5-methylthiophenyl)-N′-(3-methylthiophenyl)-N′-methylguanidine (CNS 5161) is a high affinity ligand (Ki=1.87±0.25 nM) for the NMDA PCP site, which potentially can be used for functional imaging of this receptor. Herein we report the synthesis of the corresponding positron emission tomography (PET) tracer [11C]CNS 5161 by means of [11C]methylation of the desmethyl guanidine precursor. [11C]CNS 5161 was synthesized with a decay corrected radiochemical yield of 10% within 45 min after end of bombardment (EOB). The final product was prepared in a sterile saline solution suitable for clinical studies with a radiochemical purity of >96% and a specific activity of 41 GBq/mmol at time of injection. Copyright © 2005 John Wiley & Sons, Ltd.

This unit provides a protocol for performing digital gene expression profiling on the Illumina Genome Analyzer sequencing platform. Tag sequencing (Tag-seq) is an implementation of the LongSAGE protocol on the Illumina sequencing platform that increases utility while reducing both the cost and time required to generate gene expression profiles. The ultra-high-throughput sequencing capability of the Illumina platform allows the cost-effective generation of libraries containing an average of 20 million tags, a 200-fold improvement over classical LongSAGE. Tag-seq has less sequence composition bias, leading to a better representation of AT-rich tag sequences, and allows a more accurate profiling of a subset of the transcriptome characterized by AT-rich genes expressed at levels below the threshold of detection of LongSAGE (Morrissy et al., 2009).