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Sequence capture technologies, pioneered in mammalian genomes, enable the resequencing of targeted genomic regions. Most capture protocols require blocking DNA, the production of which in large quantities can prove challenging. A blocker-free, two-stage capture protocol was developed using NimbleGen arrays. The first capture depletes the library of repetitive sequences, while the second enriches for target loci. This strategy was used to resequence non-repetitive portions of an approximately 2.2 Mb chromosomal interval and a set of 43 genes dispersed in the 2.3 Gb maize genome. This approach achieved approximately 1800-3000-fold enrichment and 80-98% coverage of targeted bases. More than 2500 SNPs were identified in target genes. Low rates of false-positive SNP predictions were obtained, even in the presence of captured paralogous sequences. Importantly, it was possible to recover novel sequences from non-reference alleles. The ability to design novel repeat-subtraction and target capture arrays makes this technology accessible in any species.

Forward genetics (phenotype-driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between observable phenotype and causative genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next-generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long-range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array-based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (approximately 200 kb) from each of five noncomplementing Kit mutants (one known allele and four unknown alleles) and have successfully identified and validated a nonsynonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover causative mutations. Importantly, these data also provide a specific methodological foundation for the development of large-scale mutation detection efforts in the laboratory mouse.

The field of high throughput proteomics has spawned a number of mass spectrometry-based technologies, which enable the quantitative analysis of protein expression. One of these technologies is iTRAQ (trademarked by Applied Biosystems), which through the use of isobaric tags, enables the quantitation of up to eight complex protein samples in a single multiplexed analysis. Isobaric tagging methods are emerging as an important tool to study protein expression dynamics. In this report, we describe iTRAQPak, a free software package developed in the R statistical and visualization environment that can be applied to the analysis of 8-plex expression data. The utility of this package is demonstrated through its application to the analysis of 8-plex iTRAQ protein expression data obtained from cerebrospinal fluid samples from Alzheimer's disease subjects involved in a Phase I drug trial.

International audience; Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

PeerGAD is a web-based database-driven application that allows community-wide peer-reviewed annotation of prokaryotic genome sequences. The application was developed to support the annotation of the Pseudomonas syringae pv. tomato strain DC3000 genome sequence and is easily portable to other genome sequence annotation projects. PeerGAD incorporates several innovative design and operation features and accepts annotations pertaining to gene naming, role classification, gene translation and annotation derivation. The annotator tool in PeerGAD is built around a genome browser that offers users the ability to search and navigate the genome sequence. Because the application encourages annotation of the genome sequence directly by researchers and relies on peer review, it circumvents the need for an annotation curator while providing added value to the annotation data. Support for the Gene Ontology™ vocabulary, a structured and controlled vocabulary used in classification of gene roles, is emphasized throughout the system. Here we present the underlying concepts integral to the functionality of PeerGAD.

The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box–containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs.

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana . The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa , yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.

The Pto gene encodes a serine/threonine protein kinase that confers resistance in tomato (Lycopersicon esculentum) to Pseudomonas syringae pv tomato strains that express the type III effector protein AvrPto. Constitutive overexpression of Pto in tomato, in the absence of AvrPto, activates defense responses and confers resistance to several diverse bacterial and fungal plant pathogens. We have used a series of gene discovery and expression profiling methods to examine the effect of Pto overexpression in tomato leaves. Analysis of the tomato expressed sequence tag database and suppression subtractive hybridization identified 600 genes that were potentially differentially expressed in Pto-overexpressing tomato plants compared with a sibling line lacking Pto. By using cDNA microarrays, we verified changes in expression of many of these genes at various time points after inoculation with P. syringae pv tomato (avrPto) of the resistant Pto-overexpressing line and the susceptible sibling line. The combination of these three approaches led to the identification of 223 POR (Pto overexpression responsive) genes. Strikingly, 40% of the genes induced in the Pto-overexpressing plants previously have been shown to be differentially expressed during the human (Homo sapiens) and/or fruitfly (Drosophila melanogaster) immune responses.

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved ( i ) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn 5gus mutagenesis, ( ii ) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, ( iii ) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and ( iv ) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N ɛ -(indole-3-acetyl)- l -lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2 , hopPtoB2 , and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.

We examined the response of the widely used Folin-Denis assay to purified tannins from 16 woody plant species and to three commercial polyphenol preparations often used as standards. The reagent's response to these chemical mixtures differed significantly among sources (tree species, commercial preparations) and sampling dates, even though the mixtures contained the same total dry weight of tannins. Response to commercial standards usually did not resemble response to actual plant tannin and produced estimates that differed from actual concentrations by as much as twofold. Species-based and seasonal differences in polyphenol composition are evidently responsible for these variable results. Reagents that depend on redox reactions, such as the Folin-Denis, do not produce reliable absolute or relative quantification of phenolics when different species or samples from different dates are compared, and use of commercial standards does not resolve this problem.

The tomato Pto kinase confers resistance to Pseudomonas syringae pv. tomato expressing the AvrPto protein. To elucidate the role of Pto autophosphorylation in disease resistance, eight sites autophosphorylated by Pto in vitro were identified by a combination of HPLC purification of tryptic phosphopeptides, MALDI-TOF/MS analysis and Edman degradation. Mutational analysis of the autophosphorylation sites revealed that Pto residues Thr38 and Ser198 are required for AvrPto-Pto- mediated elicitation of a hypersensitive response in the plant. Thr38, which is the main Pto autophosporylation site and is located outside the kinase catalytic domain, was also required for Pto kinase activity and its physical interaction with AvrPto, the Pti1 kinase and the transcription factor Pti4. Ser198, located in the Pto activation domain, was dispensable for kinase activity and for interaction with AvrPto. However, a mutation at this site resulted in altered Pto interactions with the Pti1 kinase and the Pto interactors of unknown function Pti3 and Pti10. These results suggest that autophosphorylation events at Pto Thr38 and Ser198 are required for signal transduction by Pto and participate in distinct molecular mechanisms.

The Pto and Pti1 serine/threonine protein kinases are key components of the signaling pathway leading to speck disease resistance in tomato. The two kinases physically interact in the yeast two-hybrid system, and Pto specifically phosphorylates Pti1 in vitro. In this study, we identified and characterized the major Pti1 site phosphorylated by Pto. Pto was expressed in Escherichia coli as a maltose-binding fusion protein (MBP-Pto), and used to phosphorylate in vitro a kinase deficient Pti1 protein fused to glutathione S-transferase (GST-Pti1[K96N]). The major phosphopeptide derived from trypsin digestion of phosphorylated GST-Pti1(K96N) was partially purified by reverse-phase HPLC and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Its mass corresponded to phosphopeptide LHSTR, which lies in the Pti1 kinase activation domain at amino acid position 230–234. By phosphoamino acid analysis, Thr233 was determined to be the phosphorylation site of peptide LHSTR. Mutations of Thr233 reduced dramatically Pti1 phosphorylation by MBP-Pto and Pti1 autophosphorylation, providing evidence that the same Pti1 site is involved in the two reactions. Moreover, phosphorylation of Thr233 appeared to be required for Pto–Pti1 physical interaction, as a mutation of this site to alanine, but not to aspartate, abolished the interaction between Pto and Pti1 in the yeast two-hybrid system.

Next generation sequencing (NGS) of enriched targets is increasingly being used to discover and track mutations in genes implicated in cancer. Amplicon-based target enrichment approaches are frequently used with cancer samples because there is no sample library preparation needed and primer-based approaches are typically efficient with sequencing reads. However, traditional amplicon approaches don't scale up well and may have PCR and coverage uniformity bias. As the number of samples is increased, a fast, reliable, and cost-effective target enrichment method is a critical tool for mutation discovery and tracking. Existing target enrichment approaches based on molecular inversion probes (MIPs) have presented challenges with probe design and dependability. We have developed HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) using advanced versions of MIPs which enable library-free enrichment, improved scalability, and the ability to remove PCR duplicates. We have also developed companion command line software which trims primer sequences and removes PCR duplicates. This software uses community standard file formats (FASTQ, SAM/BAM) and is intended to be compatible with common “best practice” NGS analysis pipelines. Here we describe two HEAT-Seq cancer panels which have been optimized to improve coverage uniformity and probe reliability. The HEAT-Seq Oncology Panel targets both strands for all protein coding bases in 60 cancer-related genes where possible. The target for this design is 245 kb. With 2 million 2×76bp reads for each of 8 replicates, we observe an on-target read rate of >95%. After duplicate removal, the% of target bases with at least 20x coverage is >90%. We observe uniformity (percent of probes > = 20% of the target mean) of ∼90%. The HEAT-Seq Ultra Hot-Spot Panel is a mutation-focused design that provides ultra-deep sequencing coverage capability for sequencing of mutation hot-spots in 53 cancer-related genes to detect low frequency variants in heterogeneous samples. The target for this design is 30.5 kb. With 500,000 2×76bp reads for each of 24 replicates, we observe an on-target read rate of ∼80%. After duplicate removal, the% of target bases with at least 20x coverage is >95%. We observe uniformity (percent of probes > = 20% of the target mean) of ∼95%. We also observed detection of validated low frequency variants down to 1%. In summary, both panels have been shown to capture cancer-related genes and target regions at depths sufficient to identify and track genomic variants. Citation Format: Ryan Bannen, Michael Brockman, Mark D’Ascenzo, Keynttisha Jefferson, Dawn Green, Heather Halvensleben, Kurt Heilman, Todd Richmond, Daniel Burgess. Cancer target enrichment panels using advanced molecular inversion probes (MIPs) with ability to reduce amplification bias and detect low frequency variants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5217.

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.

Sequence capture technologies, pioneered in mammalian genomes, enable the resequencing of targeted genomic regions. Most capture protocols require blocking DNA, the production of which in large quantities can prove challenging. A blocker-free, two-stage capture protocol was developed using NimbleGen arrays. The first capture depletes the library of repetitive sequences, while the second enriches for target loci. This strategy was used to resequence non-repetitive portions of an approximately 2.2 Mb chromosomal interval and a set of 43 genes dispersed in the 2.3 Gb maize genome. This approach achieved approximately 1800-3000-fold enrichment and 80-98% coverage of targeted bases. More than 2500 SNPs were identified in target genes. Low rates of false-positive SNP predictions were obtained, even in the presence of captured paralogous sequences. Importantly, it was possible to recover novel sequences from non-reference alleles. The ability to design novel repeat-subtraction and target capture arrays makes this technology accessible in any species.

An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of 8 proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2-plex and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid proteins from subjects undergoing intravenous immunoglobulin treatment for Alzheimer’s disease. Results from this study identify a number of protein expression changes that occur in cerebrospinal fluid after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.

There is significant interest in the identification of effective biomarkers for Alzheimer's disease. Such biomarkers could aid in the clinical diagnosis of the disease and may be useful in assessing the efficacy of various treatment strategies. The search for biomarkers often includes the analysis of changes in cerebrospinal fluid protein expression that correlate with disease. These changes can be measured using a variety of technologies for protein expression profiling. Although there is great promise in the application of these methods to biomarker discovery based on some preliminary observations, there are significant issues in the capabilities of most of these technologies that have limited their effective application. The most recent literature involving proteomic discovery of new cerebrospinal fluid biomarkers for Alzheimer's disease is reviewed.

In tomato plants, the Pto kinase confers resistance to the bacterial speck pathogen, Pseudomonas syringae pv. tomato, that expresses the avirulence gene avrPto. The resistant phenotype is the final result of a phosphorylation cascade triggered by the specific recognition between the AvrPto signal molecule and the Pto kinase, which is thought to function as an intracellular receptor. As a first step in the investigation of the molecular mechanisms leading to Pto activation, we characterized in vitro the biochemical properties of Pto autophosphorylation and phosphorylation of its putative effector Pti1. Pto autophosphorylated at multiple sites, as determined by phosphopeptide mapping. In addition, Pto autophosphorylation occurred via an intramolecular mechanism, as the specific activity of the kinase was not affected by its molar concentration in the assay. Kinetic analysis of Pti1 phosphorylation by Pto revealed that this reaction proceeds with a K m of 4.1 µM and V max of 0.55 nmole/min/mg. Finally, Pto phosphorylated Pti1 at one major and at several minor sites.

In tomato plants, resistance to bacterial speck disease is mediated by a phosphorylation cascade, which is triggered by the specific recognition between the plant serine/threonine protein kinase Pto and the bacterial AvrPto protein. In the present study, we investigated in vitro biochemical properties of Pto, which appears to function as an intracellular receptor for the AvrPto signal molecule. Pto and its downstream effector Pti1, which is also a serine/threonine protein kinase, were expressed in Escherichia coli as maltose-binding protein and glutathione S-transferase fusion proteins, respectively. The two kinases each autophosphorylated at multiple sites as determined by phosphopeptide mapping. In addition, Pto and Pti1 autophosphorylation occurred via an intramolecular mechanism, as their specific activity was not affected by their molar concentration in the assay. Moreover, an active glutathione S-transferase-Pto fusion failed to phosphorylate an inactive maltose-binding protein-Pto(K69Q) fusion excluding an intermolecular mechanism of phosphorylation for Pto. Pti1 phosphorylation by Pto was also characterized and found to occur with a Km of 4.1 microM at sites similar to those autophosphorylated by Pti1. Pto and the product of the recessive allele pto phosphorylated Pti1 at similar sites, as observed by phosphopeptide mapping. This suggests that the inability of the kinase pto to confer resistance to bacterial speck disease in tomato is not caused by altered recognition specificity for Pti1 phosphorylation sites.

We have developed a solution-based method for targeted DNA capture-sequencing that is directed to the complete human exome. Using this approach allows the discovery of greater than 95% of all expected heterozygous singe base variants, requires as little as 3 Gbp of raw sequence data and constitutes an effective tool for identifying rare coding alleles in large scale genomic studies.