Next-generation sequencing applications for wheat crop improvement

? Bread wheat (Triticum aestivum; Poaceae) is a crop plant of great importance. It provides nearly 20% of the world's daily food supply measured by calorie intake, similar to that provided by rice. The yield of wheat has doubled over the last 40 years due to a combination of advanced agronomic practice and improved germplasm through selective breeding. More recently, yield growth has been less dramatic, and a significant improvement in wheat production will be required if demand from the growing human population is to be met. ? Next-generation sequencing (NGS) technologies are revolutionizing biology and can be applied to address critical issues in plant biology. Technologies can produce draft sequences of genomes with a significant reduction to the cost and timeframe of traditional technologies. In addition, NGS technologies can be used to assess gene structure and expression, and importantly, to identify heritable genome variation underlying important agronomic traits. ? This review provides an overview of the wheat genome and NGS technologies, details some of the problems in applying NGS technology to wheat, and describes how NGS technologies are starting to impact wheat crop improvement.     

The evolving landscape in the therapy of acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of myeloid precursors arrested in their maturation, creating a diverse disease entity with a wide range of responses to historically standard treatment approaches. While signifi cant progress has been made in characterizing and individualizing the disease at diagnosis to optimally inform those affected, progress in treatment to reduce relapse and induce remission has been limited thus far. In addition to a brief summary of the factors that shape prognostication at diagnosis, this review attempts to expand on the current therapies under investigation that have shown promise in treating AML, including hypomethylating agents, gemtuzumab ozogamicin, FLT3 tyrosine kinase inhibitors, antisense oligonucleotides, and other novel therapies, including aurora kinases, mTOR and PI3 kinase inhibitors, PIM kinase inhibitors, HDAC inhibitors, and IDH targeted therapies. With these, and undoubtedly many others in the future, it is the hope that by combining more accurate prognostication with more effective therapies, patients will begin to have a different, and more complete, outlook on their disease that allows for safer and more successful treatment strategies.     

Next-generation sequencing and genome evolution in allopolyploids

?Premise of the study: Hybridization and polyploidization (allopolyploidy) are ubiquitous in the evolution of plants, but tracing the origins and subsequent evolution of the constituent genomes of allopolyploids has been challenging. Genome doubling greatly complicates genetic analyses, and this has long hindered investigation in that most allopolyploid species are "nonmodel" organisms. However, recent advances in sequencing and genomics technologies now provide unprecedented opportunities to analyze numerous genetic markers in multiple individuals in any organism. ?Methods: Here we review the application of next-generation sequencing technologies to the study of three aspects of allopolyploid genome evolution: duplicated gene loss and expression in two recently formed Tragopogon allopolyploids, intergenomic interactions and chromosomal evolution in Tragopogon miscellus, and repetitive DNA evolution in Nicotiana allopolyploids. ?Key results: For the first time, we can explore on a genomic scale the evolutionary processes that are ongoing in natural allopolyploids and not be restricted to well-studied crops and genetic models. ?Conclusions: These approaches can be easily and inexpensively applied to many other plant species-making any evolutionarily provocative system a new "model" system.     

高通量测序技术及其在微生物学研究中的应用

20世纪70年代发明的核酸测序技术为基因组学及其相关学科的发展做出了巨大贡献,本世纪初发展的以Illumina公司的HiSeq 2000,ABI公司的SOLID,和Roche公司的454技术为代表的高通量测序技术又为基因组学的发展注入了新活力.本文在阐述这些技术的基础上,着重讨论了新一代测序技术在微生物领域中的应用.     

Squamousness: Next-generation sequencing reveals shared molecular features across squamous tumor types

In order to gain a better understanding of the underlying biology of squamous cell carcinoma (SCC), we tested the hypothesis that SCC originating from different organs may possess common molecular alterations. SCC samples (N = 361) were examined using clinical-grade targeted next-generation sequencing (NGS). The most frequent SCC tumor types were head and neck, lung, cutaneous, gastrointestinal and gynecologic cancers. The most common gene alterations were TP53 (64.5% of patients), PIK3CA (28.5%), CDKN2A (24.4%), SOX2 (17.7%), and CCND1 (15.8%). By comparing NGS results of our SCC cohort to a non-SCC cohort (N = 277), we found that CDKN2A, SOX2, NOTCH1, TP53, PIK3CA, CCND1, and FBXW7 were significantly more frequently altered, unlike KRAS, which was less frequently altered in SCC specimens (all P < 0.05; multivariable analysis). Therefore, we identified “squamousness” gene signatures (TP53, PIK3CA, CCND1, CDKN2A, SOX2, NOTCH 1, and FBXW7 aberrations, and absence of KRAS alterations) that were significantly more frequent in SCC versus non-SCC histologies. A multivariable co-alteration analysis established 2 SCC subgroups: (i) patients in whom TP53 and cyclin pathway (CDKN2A and CCND1) alterations strongly correlated but in whom PIK3CA aberrations were less frequent; and (ii) patients with PIK3CA alterations in whom TP53 mutations were less frequent (all P ≤ 0 .001, multivariable analysis). In conclusion, we identified a set of 8 genes altered with significantly different frequencies when SCC and non-SCC were compared, suggesting the existence of patterns for “squamousness.” Targeting the PI3K-AKT-mTOR and/or cyclin pathway components in SCC may be warranted.     

Familial long-read sequencing increases yield of de novo mutations

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Next-generation sequencing as a tool to quickly identify causative EMS-generated mutations

The advent of next generation sequencing has influenced every aspect of biological research. Many labs are now using whole genome sequencing in Arabidopsis thaliana as a means to quickly identify EMS-generated mutations present in isolated mutants. Following identification of these mutations, examination of T-DNA insertional alleles defective in candidate genes or complementation of the mutant phenotype with a wild type copy of candidate genes can be used to verify which mutation is causative for the phenotype of interest. Here, we discuss the benefits and pitfalls of using this method to identify mutations underlying phenotypes.     

Next-generation sequencing for identifying new genes in rare genetic diseases: many challenges and a pinch of luck

A report on the European Society of Human Genetics conference, held in Paris, France, June 8-11, 2013.     

Current challenges in de novo plant genome sequencing and assembly

Genome sequencing is now affordable, but assembling plant genomes de novo remains challenging. We assess the state of the art of assembly and review the best practices for the community.     

Hepatosplenic candidiasis after neutropenic phase of acute leukaemia

Hepatosplenic candidiasis following granulocytopenic periods is a relatively recently recognised problem in immunocompromised patients, particularly in those with acute leukaemia. We present three patients in whom diagnosis of hepatosplenic candidiasis was suspected on the basis of ultrasonographic (US), computed tomographic (CT) findings and confirmed by laparoscopy and biopsy of liver lesions. All three patients were successfully treated briefly with amphotericin B, followed by a longer period of fluconazole. In one patient laparotomy and surgical evacuation of abscesses was performed. This condition could be more often recognised by careful follow-up of liver function test, C-reactive protein level, ultrasonography, CT and MRI after recovery from chemotherapy-induced neutropenia.     

MicroRNA control of myelopoiesis and the differentiation block in acute myeloid leukaemia

In the relatively short period of time since their discovery, microRNAs have been shown to control many important cellular functions such as cell differentiation, growth, proliferation and apoptosis. In addition, microRNAs have been demonstrated as key drivers of many malignancies and can function as either tumour suppressors or oncogenes. The haematopoietic system is not outside the realm of microRNA control with microRNAs controlling aspects of stem cell and progenitor self-renewal and differentiation, with many, if not all, haematological disorders associated with aberrant microRNA expression and function. In this review, we focus on the current understanding of microRNA control of haematopoiesis and detail the evidence for the contribution and clinical relevance of aberrant microRNA function to the characteristic block of differentiation in acute myeloid leukaemia.     

On peptide de novo sequencing: a new approach.

A procedure is presented for the automatic determination of the amino acid sequence of peptides by processing data obtained from mass spectrometry analysis. This is a basic and relevant problem in the field of proteomics. Furthermore, it has an even higher conceptual and applicative interest in peptide research, as well as in other connected fields. The analysis does not rely on known protein databases, but on the computation of all amino acid sequences compatible with the given spectral data. By formulating a mathematical model for such combinatorial problems, the structural limitations of known methods are overcome, and efficient solution algorithms can be developed. The results are very encouraging both from the accuracy and computational points of view.     

Unfavourable clinical implications for HLA-G expression in acute myeloid leukaemia

Human leukocyte antigen-G (HLA-G) molecule exerts multiple immunoregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Studies on HLA-G expression in malignant haematopoietic diseases are controversial, and the functions of HLA-G on this context are limited. In the current study, HLA-G expression was analysed in different types of patients: de novo acute myeloid leukaemia (AML, n = 54), B cell acute lymphoblastic leukaemia (B-ALL, n= 13), chronic myeloid leukaemia (CML, n= 9) and myelodysplastic syndrome (MDS, n= 11). HLA-G expression was observed in 18.5% cases of AML, 22.2% in CML and 18.2% in MDS, but not in B-ALL patients. In AML, HLA-G-positive patients had a significant higher bone marrow leukaemic blast cell percentage when compared with that of HLA-G-negative patients (P < 0.01). Total T-cell percentage was dramatically decreased in HLA-G-positive patients (P < 0.05). Cytogenetic karyotyping results showed that all HLA-G-positive AML patients (n= 5) were cytogenetically abnormal, which was markedly different from that of HLA-G-negative patients (P < 0.01). Ex vivo cytotoxicity analysis revealed that HLA-G expression in AML leukaemic cells could directly inhibit NK cell cytolysis (P < 0.01). These findings indicated that HLA-G expression in AML is of unfavourable clinical implications, and that HLA-G could be a potential target for therapy.     

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara‐C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara‐C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara‐C were elucidated by whole‐exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara‐C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.     

Assessing peptide de novo sequencing algorithms performance on large and diverse data sets

De novo peptide sequencing algorithms are often tested on relatively small data sets made of excellent spectra. Since there are always more and more tandem mass spectra available, we have assembled six large, reliable, and diverse (three mass spectrometer types) data sets intended for such tests and we make them accessible via a web server. To exemplify their use we investigate the performance of Lutefisk, PepNovo, and PepNovoTag, three well-established peptide de novo sequencing programs.     

Establishment of a prognostic ferroptosis-related gene profile in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a heterogeneous disease with a difficult to predict prognosis. Ferroptosis, an iron-induced programmed cell death, is a promising target for cancer therapy. Nevertheless, not much is known about the relationship between ferroptosis-related genes and AML prognosis. Herein, we retrieved RNA profile and corresponding clinical data of AML patients from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Univariate Cox analysis was employed to identify ferroptosis-related genes significantly associated with AML prognosis. Next, the least absolute shrinkage and selection operator (LASSO) regression was employed to establish a prognostic ferroptosis-related gene profile. 12 ferroptosis-related genes were screened to generate a prognostic model, which stratified patients into a low- (LR) or high-risk (HR) group. Using Kaplan-Meier analysis, we demonstrated that the LR patients exhibited better prognosis than HR patients. Moreover, receiver operating characteristic (ROC) curve analysis confirmed that the prognostic model showed good predictability. Functional enrichment analysis indicated that the infiltration of regulatory T cells (Treg) differed vastly between the LR and HR groups. Our prognostic model can offer guidance into the accurate prediction of AML prognosis and selection of personalized therapy in clinical practice.     

C-terminal de novo sequencing of peptides using oxazolone-based derivatization with bromine signature

Due to almost identical chemical properties of C-terminal and side-chain carboxylic groups, selective C-terminal derivatization has been difficult. Although oxazolone-based C-terminal derivatization is the only selective C-terminal modification available, it has not been used widely because of its low derivatization efficiency. In this paper, an improved oxazolone chemistry for incorporation of Br signature to C-terminus is reported. MS/MS analysis of the brominated peptides led to a series of y ions with Br signature, facilitating de novo C-terminal sequencing.     

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics

? Premise of the study: Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. ? Methods: Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). ? Key results: Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. ? Conclusions: Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.