Harm Nijveen

Primer3Plus, an enhanced web interface to Primer3

Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/primer3plus.

The Human Leukocyte Antigen–presented Ligandome of B Lymphocytes

Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action and in cellular immunotherapy and transplantation. In this paper we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA ligandome of B cells. This large number of identified ligands provides general insight into the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule, and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands, and it can be a good starting point for solving a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes based not on in silico predictions but on the bona fide eluted HLA ligandome.

Interaction between parental environment and genotype affects plant and seed performance in Arabidopsis

Highlight text The genotype-by-environment interactions of five parental environments with seed and plant performance are mediated by distinct genetic and molecular pathways, and the selective pressures that have shaped their natural variation.

Learning from co-expression networks : Possibilities and challenges

Plants are fascinating and complex organisms. A comprehensive understanding of the organization, function and evolution of plant genes is essential to disentangle important biological processes and to advance crop engineering and breeding strategies. The ultimate aim in deciphering complex biological processes is the discovery of causal genes and regulatory mechanisms controlling these processes. The recent surge of omics data has opened the door to a system-wide understanding of the flow of biological information underlying complex traits. However, dealing with the corresponding large data sets represents a challenging endeavor that calls for the development of powerful bioinformatics methods. A popular approach is the construction and analysis of gene networks. Such networks are often used for genome-wide representation of the complex functional organization of biological systems. Network based on similarity in gene expression are called (gene) co-expression networks. One of the major application of gene co-expression networks is the functional annotation of unknown genes. Constructing co-expression networks is generally straightforward. In contrast, the resulting network of connected genes can become very complex, which limits its biological interpretation. Several strategies can be employed to enhance the interpretation of the networks. A strategy in coherence with the biological question addressed needs to be established to infer reliable networks. Additional benefits can be gained from network-based strategies using prior knowledge and data integration to further enhance the elucidation of gene regulatory relationships. As a result, biological networks provide many more applications beyond the simple visualization of co-expressed genes. In this study we review the different approaches for co-expression network inference in plants. We analyse integrative genomics strategies used in recent studies that successfully identified candidate genes taking advantage of gene co-expression networks. Additionally, we discuss promising bioinformatics approaches that predict networks for specific purposes.

Remarkably Divergent Regions Punctuate the Genome Assembly of the Caenorhabditis elegans Hawaiian Strain CB4856

The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population, and evolutionary studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. When compared against the N2 reference, the CB4856 genome has 327,050 single nucleotide variants (SNVs) and 79,529 insertion–deletion events that result in a total of 3.3 Mb of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 but not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, which have a greatly elevated SNV density, ranging from 2 to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors, suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes.

Understanding and identifying amino acid repeats

Amino acid repeats (AARs) are abundant in protein sequences. They have particular roles in protein function and evolution. Simple repeat patterns generated by DNA slippage tend to introduce length variations and point mutations in repeat regions. Loss of normal and gain of abnormal function owing to their variable length are potential risks leading to diseases. Repeats with complex patterns mostly refer to the functional domain repeats, such as the well-known leucine-rich repeat and WD repeat, which are frequently involved in protein–protein interaction. They are mainly derived from internal gene duplication events and stabilized by ‘gate-keeper’ residues, which play crucial roles in preventing inter-domain aggregation. AARs are widely distributed in different proteomes across a variety of taxonomic ranges, and especially abundant in eukaryotic proteins. However, their specific evolutionary and functional scenarios are still poorly understood. Identifying AARs in protein sequences is the first step for the further investigation of their biological function and evolutionary mechanism. In principle, this is an NP-hard problem, as most of the repeat fragments are shaped by a series of sophisticated evolutionary events and become latent periodical patterns. It is not possible to define a uniform criterion for detecting and verifying various repeat patterns. Instead, different algorithms based on different strategies have been developed to cope with different repeat patterns. In this review, we attempt to describe the amino acid repeat-detection algorithms currently available and compare their strategies based on an in-depth analysis of the biological significance of protein repeats.

A footprint of desiccation tolerance in the genome of Xerophyta viscosa

Desiccation tolerance is common in seeds and various other organisms, but only a few angiosperm species possess vegetative desiccation tolerance. These ‘resurrection species’ may serve as ideal models for the ultimate design of crops with enhanced drought tolerance. To understand the molecular and genetic mechanisms enabling vegetative desiccation tolerance, we produced a high-quality whole-genome sequence for the resurrection plant Xerophyta viscosa and assessed transcriptome changes during its dehydration. Data revealed induction of transcripts typically associated with desiccation tolerance in seeds and involvement of orthologues of ABI3 and ABI5, both key regulators of seed maturation. Dehydration resulted in both increased, but predominantly reduced, transcript abundance of genomic ‘clusters of desiccation-associated genes’ (CoDAGs), reflecting the cessation of growth that allows for the expression of desiccation tolerance. Vegetative desiccation tolerance in X. viscosa was found to be uncoupled from drought-induced senescence. We provide strong support for the hypothesis that vegetative desiccation tolerance arose by redirection of genetic information from desiccation-tolerant seeds.

Beyond genomic variation - comparison and functional annotation of three Brassica rapa genomes: a turnip, a rapid cycling and a Chinese cabbage

BackgroundBrassica rapa is an economically important crop species. During its long breeding history, a large number of morphotypes have been generated, including leafy vegetables such as Chinese cabbage and pakchoi, turnip tuber crops and oil crops.ResultsTo investigate the genetic variation underlying this morphological variation, we re-sequenced, assembled and annotated the genomes of two B. rapa subspecies, turnip crops (turnip) and a rapid cycling. We then analysed the two resulting genomes together with the Chinese cabbage Chiifu reference genome to obtain an impression of the B. rapa pan-genome. The number of genes with protein-coding changes between the three genotypes was lower than that among different accessions of Arabidopsis thaliana, which can be explained by the smaller effective population size of B. rapa due to its domestication. Based on orthology to a number of non-brassica species, we estimated the date of divergence among the three B. rapa morphotypes at approximately 250,000 YA, far predating Brassica domestication (5,000-10,000 YA).ConclusionsBy analysing genes unique to turnip we found evidence for copy number differences in peroxidases, pointing to a role for the phenylpropanoid biosynthesis pathway in the generation of morphological variation. The estimated date of divergence among three B. rapa morphotypes implies that prior to domestication there was already considerably divergence among B. rapa genotypes. Our study thus provides two new B. rapa reference genomes, delivers a set of computer tools to analyse the resulting pan-genome and uses these to shed light on genetic drivers behind the rich morphological variation found in B. rapa.

QualitySNPng: a user-friendly SNP detection and visualization tool

QualitySNPng is a new software tool for the detection and interactive visualization of single-nucleotide polymorphisms (SNPs). It uses a haplotype-based strategy to identify reliable SNPs; it is optimized for the analysis of current RNA-seq data; but it can also be used on genomic DNA sequences derived from next-generation sequencing experiments. QualitySNPng does not require a sequenced reference genome and delivers reliable SNPs for di- as well as polyploid species. The tool features a user-friendly interface, multiple filtering options to handle typical sequencing errors, support for SAM and ACE files and interactive visualization. QualitySNPng produces high-quality SNP information that can be used directly in genotyping by sequencing approaches for application in QTL and genome-wide association mapping as well as to populate SNP arrays. The software can be used as a stand-alone application with a graphical user interface or as part of a pipeline system like Galaxy. Versions for Windows, Mac OS X and Linux, as well as the source code, are available from http://www.bioinformatics.nl/QualitySNPng.

A gene co-expression network predicts functional genes controlling the re-establishment of desiccation tolerance in germinated Arabidopsis thaliana seeds

AbstractMain conclusionDuring re-establishment of desiccation tolerance (DT), early events promote initial protection and growth arrest, while late events promote stress adaptation and contribute to survival in the dry state. Mature seeds of Arabidopsis thaliana are desiccation tolerant, but they lose desiccation tolerance (DT) while progressing to germination. Yet, there is a small developmental window during which DT can be rescued by treatment with abscisic acid (ABA). To gain temporal resolution and identify relevant genes in this process, data from a time series of microarrays were used to build a gene co-expression network. The network has two regions, namely early response (ER) and late response (LR). Genes in the ER region are related to biological processes, such as dormancy, acquisition of DT and drought, amplification of signals, growth arrest and induction of protection mechanisms (such as LEA proteins). Genes in the LR region lead to inhibition of photosynthesis and primary metabolism, promote adaptation to stress conditions and contribute to seed longevity. Phenotyping of 12 hubs in relation to re-establishment of DT with T-DNA insertion lines indicated a significant increase in the ability to re-establish DT compared with the wild-type in the lines cbsx4, at3g53040 and at4g25580, suggesting the operation of redundant and compensatory mechanisms. Moreover, we show that re-establishment of DT by polyethylene glycol and ABA occurs through partially overlapping mechanisms. Our data confirm that co-expression network analysis is a valid approach to examine data from time series of transcriptome analysis, as it provides promising insights into biologically relevant relations that help to generate new information about the roles of certain genes for DT.