Methods in molecular biology
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In the past decade, proteomics and mass spectrometry have taken tremendous strides forward, particularly in the life sciences, spurred on by rapid advances in technology resulting in generation and conglomeration of vast amounts of data. Though this has led to tremendous advancements in biology, the interpretation of the data poses serious challenges for many practitioners due to the immense size and complexity of the data. Furthermore, the lack of annotation means that a potential gold mine of relevant biological information may be hiding within this data. ⋯ We then integrate a suite of freely available bioinformatics analysis and annotation software tools to identify homologues and map putative functional signatures, gene ontology and biochemical pathways. We also provide an example of the functional annotation of missing proteins in human chromosome 7 data from the NeXtProt database, where no evidence is available at the proteomic, antibody, or structural levels. We give examples of protocols, tools and detailed flowcharts that can be extended or tailored to interpret and annotate the proteome of any novel organism.
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DNA methylation is a major epigenetic modification that regulates gene expression, genome imprinting, and development and has a role in diseases including cancer. There are various methods for whole-genome methylation profiling that differ in cost and resolution. ⋯ In this chapter, we provide detailed protocols for whole-genome bisulfite sequencing (WGBS), which captures the complete methylome. Using WGBS, we are able to generate a reference DNA methylome for normal or malignant hematopoietic cells.
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The activated partial thromboplastin time (APTT) is a useful global assay for the assessment of the contact factor pathway of hemostasis and its inhibitors. The test is usually performed on fully automated analyzers using commercially prepared reagents. The three main clinical areas of interest are detection of factor deficiencies, detection of lupus anticoagulants and in the monitoring of therapy with unfractionated heparin. Methods are described here for assessing APTT reagents for their sensitivity to clotting time prolongation in each of these areas of interest.
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Network analysis methods are increasing in popularity. An approach commonly applied to analyze proteomics data involves the use of protein-protein interaction (PPI) networks to explore the systems-level cooperation between proteins identified in a study. ⋯ Here we describe a method for calculating robust empirical p-values for protein interaction networks. We also provide a worked example with python code demonstrating the implementation of this methodology.
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Whole-genome bisulfite sequencing (WGBS) has become a powerful tool to dissect genome-wide methylation profiles at single-base resolution. In this chapter we describe in detail the bioinformatics pipeline used for the analysis of ARGONAUTE-dependent DNA methylation in Arabidopsis thaliana. We provide tools and command lines used for mapping bisulfite sequencing reads, for estimating methylation levels at individual cytosine sites, for identifying differentially methylated regions (DMRs), and for calculating methylation levels of DMRs.