Alternate wetting and drying irrigation for rice in Bangladesh: Is it sustainable and has plant breeding something to offer?

The crop management practice of alternate wetting and drying (AWD) is being promoted by IRRI and the national research and extension program in Bangladesh and other parts of the world as a water-saving irrigation practice that reduces the environmental impact of dry season rice production through decreased water usage, and potentially increases yield. Evidence is growing that AWD will dramatically reduce the concentration of arsenic in harvested rice grains conferring a third major advantage over permanently flooded dry season rice production. AWD may also increase the concentration of essential dietary micronutrients in the grain. However, three crucial aspects of AWD irrigation require further investigation. First, why is yield generally altered in AWD? Second, is AWD sustainable economically (viability of farmers' livelihoods) and environmentally (aquifer water table heights) over long-term use? Third, are current cultivars optimized for this irrigation system? This paper describes a multidisciplinary research project that could be conceived which would answer these questions by combining advanced soil biogeochemistry with crop physiology, genomics, and systems biology. The description attempts to show how the breakthroughs in next generation sequencing could be exploited to better utilize local collections of germplasm and identify the molecular mechanisms underlying biological adaptation to the environment within the context of soil chemistry and plant physiology.

Arbuscular mycorrhizal fungal communities are phylogenetically clustered at small scales

Next-generation sequencing technologies with markers covering the full Glomeromycota phylum were used to uncover phylogenetic community structure of arbuscular mycorrhizal fungi (AMF) associated with Festuca brevipila. The study system was a semi-arid grassland with high plant diversity and a steep environmental gradient in pH, C, N, P and soil water content. The AMF community in roots and rhizosphere soil were analyzed separately and consisted of 74 distinct operational taxonomic units (OTUs) in total. Community-level variance partitioning showed that the role of environmental factors in determining AM species composition was marginal when controlling for spatial autocorrelation at multiple scales. Instead, phylogenetic distance and spatial distance were major correlates of AMF communities: OTUs that were more closely related (and which therefore may have similar traits) were more likely to co-occur. This pattern was insensitive to phylogenetic sampling breadth. Given the minor effects of the environment, we propose that at small scales closely related AMF positively associate through biotic factors such as plant-AMF filtering and interactions within the soil biota.

Comparison of Microbiomes from Different Niches of Upper and Lower Airways in Children and Adolescents with Cystic Fibrosis

Changes in the airway microbiome may be important in the pathophysiology of chronic lung disease in patients with cystic fibrosis. However, little is known about the microbiome in early cystic fibrosis lung disease and the relationship between the microbiomes from different niches in the upper and lower airways. Therefore, in this cross-sectional study, we examined the relationship between the microbiome in the upper (nose and throat) and lower (sputum) airways from children with cystic fibrosis using next generation sequencing. Our results demonstrate a significant difference in both α and β-diversity between the nose and the two other sampling sites. The nasal microbiome was characterized by a polymicrobial community while the throat and sputum communities were less diverse and dominated by a few operational taxonomic units. Moreover, sputum and throat microbiomes were closely related especially in patients with clinically stable lung disease. There was a high inter-individual variability in sputum samples primarily due to a decrease in evenness linked to increased abundance of potential respiratory pathogens such as Pseudomonas aeruginosa. Patients with chronic Pseudomonas aeruginosa infection exhibited a less diverse sputum microbiome. A high concordance was found between pediatric and adult sputum microbiomes except that Burkholderia was only observed in the adult cohort. These results indicate that an adult-like lower airways microbiome is established early in life and that throat swabs may be a good surrogate in clinically stable children with cystic fibrosis without chronic Pseudomonas aeruginosa infection in whom sputum sampling is often not feasible.

Computational cancer biology: education is a natural key to many locks

Background: Oncology is a field that profits tremendously from the genomic data generated by high-throughput technologies, including next-generation sequencing. However, in order to exploit, integrate, visualize and interpret such high-dimensional data efficiently, non-trivial computational and statistical analysis methods are required that need to be developed in a problem-directed manner.

Discussion: For this reason, computational cancer biology aims to fill this gap. Unfortunately, computational cancer biology is not yet fully recognized as a coequal field in oncology, leading to a delay in its maturation and, as an immediate consequence, an under-exploration of high-throughput data for translational research.

Summary: Here we argue that this imbalance, favoring 'wet lab-based activities', will be naturally rectified over time, if the next generation of scientists receives an academic education that provides a fair and competent introduction to computational biology and its manifold capabilities. Furthermore, we discuss a number of local educational provisions that can be implemented on university level to help in facilitating the process of harmonization.

The fungal microbiota of de-novo paediatric inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterised by an inappropriate chronic immune response against resident gut microbes. This may be on account of distinct changes in the gut microbiota termed as dysbiosis. The role of fungi in this altered luminal environment has been scarcely reported. We studied the fungal microbiome in de-novo paediatric IBD patients utilising next generation sequencing and compared with adult disease and normal controls. We report a distinct difference in fungal species with Ascomycota predominating in control subjects compared to Basidiomycota dominance in children with IBD, which could be as a result of altered tolerance in these patients. 

Molecular diagnostics of myeloproliferative neoplasms

Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN-specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. While many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations is considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN-associated mutations and it therefore appears likely that emerging technologies such as next-generation sequencing and digital PCR will in the future, play an increasing role in the molecular diagnosis of MPN. 

Novel tools for conservation genomics: comparing two high-throughput approaches for SNP discovery in the transcriptome of the European hake

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).

Application of SNPs for population genetics of nonmodel organisms:new opportunities and challenges

Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionizing the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an addition to the molecular ecology toolkit in nonmodel organisms, but their efficient use remains challenging. Here, we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias and the inclusion of nonneutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in nonmodel taxa, focusing specifically on some of the most commonly applied methods.

Isolation and characterization of thirty microsatellite loci for Atlantic mackerel (Scomber scombrus L.)

Atlantic mackerel (Scomber scombrus L.) is a widely distributed commercially important pelagic species. Little is known about the stock structure of this species, but it is thought to be undergoing a range extension due to environmental changes. Knowledge of the stock structure under these changing conditions is fundamental for effective management. In this paper, 30 highly polymorphic microsatellite loci developed with next generation sequencing are described. The number of alleles per locus ranged from 4 to 39 in two geographically distant populations, observed and expected heterozygosities ranged between 0. 370-0. 978 and 0. 426-0. 962, respectively. These loci are an important resource that will allow assessment of the current population genetic structure of this species, and enable monitoring of climate related changes in the species range and distribution. 

Twenty-two novel microsatellite loci for lumpfish (Cyclopterus lumpus)

Lumpfish (Cyclopterus lumpus L. 1758) are widely distributed on both sides of the North Atlantic. They are a commercially important species, but stock size estimates have declined since the mid-1980s in Canada, Norway and Iceland. Little is known about the biology of this species, in particular the breeding migrations and population structure which are fundamental for effective management. This paper describes the development and characterization of twenty-two polymorphic microsatellite loci using next generation sequencing. The number of alleles per locus ranged from 3 to 27 in two geographically distant North Atlantic populations, with observed and expected heterozygosities ranging between 0. 0625-0. 979 and 0. 0618-0. 946, respectively. These loci are an important resource that will allow assessment of the population genetic structure of this species, and contribute to its appropriate management.

Multiplexing with three-primer PCR for rapid and economical microsatellite validation.

The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n 46) and the Scotian Shelf (n 46), two locations that have shown genetic differentiation in previous studies. Multilocus FST between the two samples was estimated at 0.067 (P 0.001). After three loci potentially under selection were excluded, the global FST was estimated at 0.043 (P 0.001). Our technique combines three- primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci.

Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility

Small bowel accounts for only 0.5% of cancer cases in the US but incidence rates have been rising at 2.4% per year over the past decade. One-third of these are adenocarcinomas but little is known about their molecular pathology and no molecular markers are available for clinical use. Using a retrospective 28 patient matched normal-tumor cohort, next-generation sequencing, gene expression arrays and CpG methylation arrays were used for molecular profiling. Next-generation sequencing identified novel mutations in IDH1, CDH1, KIT, FGFR2, FLT3, NPM1, PTEN, MET, AKT1, RET, NOTCH1 and ERBB4. Array data revealed 17% of CpGs and 5% of RNA transcripts assayed to be differentially methylated and expressed respectively (p < 0.01). Merging gene expression and DNA methylation data revealed CHN2 as consistently hypermethylated and downregulated in this disease (Spearman -0.71, p < 0.001). Mutations in TP53 which were found in more than half of the cohort (15/28) and Kazald1 hypomethylation were both were indicative of poor survival (p = 0.03, HR = 3.2 and p = 0.01, HR = 4.9 respectively). By integrating high-throughput mutational, gene expression and DNA methylation data, this study reveals for the first time the distinct molecular profile of small bowel adenocarcinoma and highlights potential clinically exploitable markers.

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib.

METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies.

RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib.

CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

Considerations for optimization of microRNA PCR assays for molecular diagnosis

The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.

Is it time for standards and a common data exchange format for digital image analysis?

Digital image analysis is at a crossroads. While the technology has made great strides over the past few decades, there is an urgent need for image analysis to inform the next wave of large scale tissue biomarker discovery studies in cancer. Drawing parallels from the growth of next generation sequencing, this presentation will consider the case for a common language or standard format for storing and communicating digital image analysis data. In this context, image analysis data comprises more than simply an image with markups and attached key-value pair metrics. The desire to objectively benchmark competing platforms or a push for data to be deposited to public repositories much like genomics data may drive the need for a standard that also encompasses granular, cell-by-cell data.