NGS QC Toolkit: a toolkit for quality control of next generation sequencing data

Next generation sequencing (NGS) technologies provide a high-throughput means to generate large amount of sequence data. However, quality control (QC) of sequence data generated from these technologies is extremely important for meaningful downstream analysis. Further, highly efficient and fast processing tools are required to handle the large volume of datasets. Here, we have developed an application, NGS QC Toolkit, for quality check and filtering of high-quality data. This toolkit is a standalone and open source application freely available at http://www.nipgr.res.in/ngsqctoolkit.html. All the tools in the application have been implemented in Perl programming language. The toolkit is comprised of user-friendly tools for QC of sequencing data generated using Roche 454 and Illumina platforms, and additional tools to aid QC (sequence format converter and trimming tools) and analysis (statistics tools). A variety of options have been provided to facilitate the QC at user-defined parameters. The toolkit is expected to be very useful for the QC of NGS data to facilitate better downstream analysis.     

Polycomb and Trithorax control genome expression by determining the alternative chromatin epigenetic states for key developmental regulators

The Polycomb (PcG) and Trithorax (TrxG) group proteins are essential for development in all multicellular organisms. Mutations of the PcG and TrxG genes act as early embryonic lethals, while their overexpression correlates with malignancies. Comparative genomic analysis showed that PcG and TrxG form a binary regulatory system that functions as an epigenetic rheostat to determine the threshold levels of extracellular signals affecting the expression levels of key developmental genes.     

Polycomb and trithorax control genome expression by determining the alternative epigenetic states of chromatin for key developmental regulators

The Polycomb (PcG) and Trithorax (TrxG) group proteins are essential for development in all multicellular organisms. Mutations of the PcG and TrxG genes act as early embryonic lethals, while their overexpression correlates with malignancies. Comparative genome analysis showed that PcG and TrxG form a binary regulatory system that functions as an epigenetic rheostat to determine the threshold levels of extracellular signals affecting the expression levels of key developmental genes.     

Symbiosis as a source of selectable epigenetic variation: taking the heat for the big guy

Evolutionary developmental biology is based on the principle that evolution arises from hereditable changes in development. Most of this new work has centred on changes in the regulatory components of the genome. However, recent studies (many of them documented in this volume) have shown that development also includes interactions between the organism and its environment. One area of interest concerns the importance of symbionts for the production of the normal range of phenotypes. Many, if not most, organisms have ‘outsourced’ some of their developmental signals to a set of symbionts that are expected to be acquired during development. Such intimate interactions between species are referred to as codevelopment, the production of a new individual through the coordinated interactions of several genotypically different species. Within the past 2 years, several research programmes have demonstrated that such codevelopmental schemes can be selected. We will focus on symbioses in coral reef cnidarians symbiosis, pea aphids and cactuses, wherein the symbiotic system provides thermotolerance for the composite organism.     

The L1 retrotransposition assay: A retrospective and toolkit

LINE1s (L1s) are a class of mammalian non-LTR (long terminal repeat) retroelements that make up nearly 20% of the human genome. Because of the difficulty of studying the mobilization of endogenous L1s, an exogenous cell culture retrotransposition assay has become integral to research in L1 biology. This assay has allowed for investigation of the mechanism and consequences of mobilization of this retroelement, both in cell lines and in whole animal models. In this paper, we outline the genesis of in vitro retrotransposition systems which led to the development of the L1 retrotransposition assay in the mid-1990s. We then provide a retrospective, describing the many uses and variations of this assay, ending with caveats and predictions for future developments. Finally, we provide detailed protocols on the application of the retrotransposition assay, including lists of constructs available in the L1 research community and cell lines in which this assay has been applied.     

PyEvolve: a toolkit for statistical modelling of molecular evolution

Background  

Examining the distribution of variation has proven an extremely profitable technique in the effort to identify sequences of biological significance. Most approaches in the field, however, evaluate only the conserved portions of sequences – ignoring the biological significance of sequence differences. A suite of sophisticated likelihood based statistical models from the field of molecular evolution provides the basis for extracting the information from the full distribution of sequence variation. The number of different problems to which phylogeny-based maximum likelihood calculations can be applied is extensive. Available software packages that can perform likelihood calculations suffer from a lack of flexibility and scalability, or employ error-prone approaches to model parameterisation.     

Developmental plasticity and developmental origins of non-communicable disease: theoretical considerations and epigenetic mechanisms

There is now evidence that developmental influences have lifelong effects on cardiovascular and metabolic function and that elements of the heritable or familial component of susceptibility to cardiovascular disease, obesity and other non-communicable diseases (NCD) can be transmitted across generations by non-genomic means. In animals the developmental environment induces altered phenotypes through genetic, physiological (especially endocrine) and epigenetic mechanisms. The latter include DNA methylation, covalent modifications of histones and non-coding RNAs. Such ‘tuning’ of phenotype has potential adaptive value and may confer Darwinian fitness advantage because it either adjusts the phenotype to current circumstances and/or attempts to match an individual’s responses to the environment predicted to be experienced later. When the phenotype is mismatched to the later environment, e.g. from inaccurate nutritional cues from the mother or placenta before birth, or from rapid environmental change through improved socio-economic conditions, risk of NCD increases. Such mechanisms are also thought to play roles in ageing and early onset of puberty, reinforcing a life-course perspective on such adaptive responses, especially the detrimental later effects of trade-offs. Epigenetic changes induced during development are highly gene-specific and function at the level of individual CpG dinucleotides in both gene promoter and intergenic regions. Evidence is accruing that endocrine or nutritional interventions during early postnatal life can reverse epigenetic and phenotypic changes induced, for example, by unbalanced maternal diet during pregnancy. Elucidation of epigenetic processes may permit perinatal identification of individuals most at risk of later NCD and enable early intervention strategies to reduce such risk.     

Alpha-synuclein sequesters Dnmt1 from the nucleus: a novel mechanism for epigenetic alterations in Lewy body diseases

DNA methylation is a major epigenetic modification that regulates gene expression. Dnmt1, the maintenance DNA methylation enzyme, is abundantly expressed in the adult brain and is mainly located in the nuclear compartment, where it has access to chromatin. Hypomethylation of CpG islands at intron 1 of the SNCA gene has recently been reported to result in overexpression of α-synuclein in Parkinson disease (PD) and related disorders. We therefore investigated the mechanisms underlying altered DNA methylation in PD and dementia with Lewy bodies (DLB). We present evidence of reduction of nuclear Dnmt1 levels in human postmortem brain samples from PD and DLB patients as well as in the brains of α-synuclein transgenic mice models. Furthermore, sequestration of Dnmt1 in the cytoplasm results in global DNA hypomethylation in human and mouse brains, involving CpG islands upstream of SNCA, SEPW1, and PRKAR2A genes. We report that association of Dnmt1 and α-synuclein might mediate aberrant subcellular localization of Dnmt1. Nuclear Dnmt1 levels were partially rescued by overexpression of Dnmt1 in neuronal cell cultures and in α-synuclein transgenic mice brains. Our results underscore a novel mechanism for epigenetic dysregulation in Lewy body diseases, which might underlie the decrease in DNA methylation reported for PD and DLB.     

Procedure control and acceptance sampling plans for donor sperm banks: a theoretical study

The publication of European Directive 2004/23/EC in the European Parliament and in the European Council on 31 March 2004 concerning the setting of standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human cells and tissues made it obligatory for sperm banks to set up quality control systems to ensure, among other goals, the satisfactory control of all procedures carried out. The objective of the present study is to set out guidelines that will make it possible to ensure the quality of the donors and frozen specimens accepted and the homogeneity of the samples supplied by a sperm bank. For this purpose, we shall describe clear-cut criteria for the acceptance of donors and frozen sperm, taking into account both analytic variability and the biological variations to be expected in semen parameters. Furthermore, we shall show how the evaluation of the results of a frozen semen specimen, on the basis of analysing a single straw after such freezing, does not guarantee the homogeneity of all the straws. Therefore, we must design a sampling plan to take into consideration all the straws obtained from a donor. This kind of plan will depend on different parameters, such as acceptable levels of quality and the tolerable rate of straws with defective semen, and will involve certain risks, both for the sperm bank and for the client. The establishment of these acceptance control criteria for frozen specimens and for donors could be of practical use for the control of the procedures applied in the operation of a sperm bank.     

组蛋白甲基转移酶G9a在表观遗传调控中的作用

组蛋白赖氨酸甲基化在表观遗传调控中起着关键作用。组蛋白甲基转移酶G9a(又称作常染色质组蛋白赖氨酸N-甲基转移酶2(euchromatic histone-lysine N-methyltransferase 2,EHMT2))含经典的SET结构域,是常染色质主要的甲基转移酶之一,可以甲基化组蛋白H3K9、H3K27和H1bK26等。此外,G9a也可以直接甲基化一些非组蛋白,并与DNA甲基化密切相关。G9a功能紊乱可以导致胚胎发育异常、免疫系统及神经系统发育障碍、甚至癌症的发生发展。     

MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity

MCScan is an algorithm able to scan multiple genomes or subgenomes in order to identify putative homologous chromosomal regions, and align these regions using genes as anchors. The MCScanX toolkit implements an adjusted MCScan algorithm for detection of synteny and collinearity that extends the original software by incorporating 14 utility programs for visualization of results and additional downstream analyses. Applications of MCScanX to several sequenced plant genomes and gene families are shown as examples. MCScanX can be used to effectively analyze chromosome structural changes, and reveal the history of gene family expansions that might contribute to the adaptation of lineages and taxa. An integrated view of various modes of gene duplication can supplement the traditional gene tree analysis in specific families. The source code and documentation of MCScanX are freely available at http://chibba.pgml.uga.edu/mcscan2/.     

Conformational states of human rat sarcoma (Ras) protein complexed with its natural ligand GTP and their role for effector interaction and GTP hydrolysis

The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH(2)p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer (31)P NMR experiments and effector binding studies we show that Ras(wt)·Mg(2+)·GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K(12) is 11.3. K(12) of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k(ex)) for Ras(wt)·Mg(2+)·GTP is 7 s(-1) and thus 18-fold lower compared with that found for the Ras·GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.     

GBSX: a toolkit for experimental design and demultiplexing genotyping by sequencing experiments

Background

Massive parallel sequencing is a powerful tool for variant discovery and genotyping. To reduce costs, sequencing of restriction enzyme based reduced representation libraries can be utilized. This technology is generally referred to as Genotyping By Sequencing (GBS). To deal with GBS experimental design and initial processing specific bioinformatic tools are needed.

Results

GBSX is a package that assists in selecting the appropriate enzyme and the design of compatible in-line barcodes. Post sequencing, it performs optimized demultiplexing using these barcodes to create fastq files per barcode which can easily be plugged into existing variant analysis pipelines. Here we demonstrate the usability of the GBSX toolkit and demonstrate improved in-line barcode demultiplexing and trimming performance compared to existing tools.

Conclusions

GBSX provides an easy to use suite of tools for designing and demultiplexing of GBS experiments.