The Potential and Challenges of Nanopore Sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of ‘third generation’ instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.Molecular and Cellular BiologyPhysic

Genomic Profiling of Childhood Tumor Patient-Derived Xenograft Models to Enable Rational Clinical Trial Design.

Accelerating cures for children with cancer remains an immediate challenge as a result of extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs)-many of which are refractory to current standard-of-care treatments-from high-risk childhood cancers. Here, we genomically characterize 261 PDX models from 37 unique pediatric cancers; demonstrate faithful recapitulation of histologies and subtypes; and refine our understanding of relapsed disease. In addition, we use expression signatures to classify tumors for TP53 and NF1 pathway inactivation. We anticipate that these data will serve as a resource for pediatric oncology drug development and will guide rational clinical trial design for children with cancer

Characterization of endoplasmic reticulum from castor bean and the cloning of a plant phosphatase: a basis for comprehensive plant organelle proteomics research

The plant endoplasmic reticulum is the location of storage oil and membrane lipid assembly, and for fatty acid modifying reactions (desaturation, elongation, hydroxylation). It therefore represents a source of enzymes involved in these processes. Many of these defy traditional purification strategies. In this study, ER membranes have been isolated biochemically pure and in milligram quanties from the endosperm of developing and germinating castor bean. One-dimensional SDS- PAGE, used to routinely assess sample integrity, showed resolution limitations. Two-dimensional gel electrophoresis was optimized regarding sample preparation and solubilization, and reproducible profiles confirmed its suitability as a sound basis for analysis of stage-specific ER components. In large format 2-D experiments, preparative loadings were reproducibly resolved. MALDI TOP mass spectrometry was evaluated for high throughput peptide signature generation with individual ER components. Resolution problems were again highlighted with 1-D separations, although some functional assignments were made. Subsequently analysis of selected spots from a preparative 2-D gel of germinating ER was used to establish the limitations of the procedure. Database matching of a single component at very low levels of mass error tolerance also demonstrated the power and accuracy of the technology. Membranes were subfractionated to simplify protein patterns. It is proposed that an organellar approach, including subfractionation, provides enrichment of specific subsets of cellular components. A putative plant phosphatidic acid phosphatase gene has been investigated following identification from the EST database. The aim of this research is the identification of proteins involved in storage lipid synthesis in castor bean in reactions specific to the endoplasmic reticulum

Comparison of Accuracy on DNA Quantitation Determined by MALDI-TOF Mass Spectrometry and UV Spectrometry

Although the UV absorbance of DNA at 260 nm has been recognized as a standard method for DNA quantitation, there are limitations of using UV spectrometry to determine the purity and identity of DNA. Recently, MALDI-TOF MS has proven to be an accurate technique for qualitative DNA analysis. In this study, the accuracy of MALDI-TOF MS for determining the concentration of DNA is evaluated and compared with that of the standard UV method. The results indicated that the accuracy of quantitative MALDI-TOF MS was comparable to that of the standard UV method and that measured DNA concentrations correlated well with those determined by the standard UV method

Mutations in <em>GRHL2</em> result in an autosomal-recessive ectodermal dysplasia syndrome

Grainyhead-like 2, encoded by GRHL2, is a member of a highly conserved family of transcription factors that play essential roles during epithelial development. Haploinsufficiency for GRHL2 has been implicated in autosomal-dominant deafness, but mutations have not yet been associated with any skin pathology. We investigated two unrelated Kuwaiti families in which a total of six individuals have had lifelong ectodermal defects. The clinical features comprised nail dystrophy or nail loss, marginal palmoplantar keratoderma, hypodontia, enamel hypoplasia, oral hyperpigmentation, and dysphagia. In addition, three individuals had sensorineural deafness, and three had bronchial asthma. Taken together, the features were consistent with an unusual autosomal-recessive ectodermal dysplasia syndrome. Because of consanguinity in both families, we used whole-exome sequencing to search for novel homozygous DNA variants and found GRHL2 mutations common to both families: affected subjects in one family were homozygous for c.1192T>C (p.Tyr398His) in exon 9, and subjects in the other family were homozygous for c.1445T>A (p.Ile482Lys) in exon 11. Immortalized keratinocytes (p.Ile482Lys) showed altered cell morphology, impaired tight junctions, adhesion defects, and cytoplasmic translocation of GRHL2. Whole-skin transcriptomic analysis (p.Ile482Lys) disclosed changes in genes implicated in networks of cell-cell and cell-matrix adhesion. Our clinical findings of an autosomal-recessive ectodermal dysplasia syndrome provide insight into the role of GRHL2 in skin development, homeostasis, and human disease

The Quest for Rare Variants: Pooled Multiplexed Next Generation Sequencing in Plants

Next generation sequencing (NGS) instruments produce an unprecedented amount of sequence data at contained costs. This gives researchers the possibility of designing studies with adequate power to identify rare variants at a fraction of the economic and labor resources required by individual Sanger sequencing. As of today, few research groups working in plant sciences have exploited this potentiality, showing that pooled NGS provides results in excellent agreement with those obtained by individual Sanger sequencing. The aim of this review is to convey to the reader the general ideas underlying the use of pooled NGS for the identification of rare variants. To facilitate a thorough understanding of the possibilities of the method, we will explain in detail the possible experimental and analytical approaches and discuss their advantages and disadvantages. We will show that information on allele frequency obtained by pooled NGS can be used to accurately compute basic population genetics indexes such as allele frequency, nucleotide diversity, and Tajima’s D. Finally, we will discuss applications and future perspectives of the multiplexed NGS approach

Mass spectrometric methods and bioinformatics tools for accurate identification of MicroRNA biomarkers

MicroRNA (miRNA) are a class of endogenous non-protein-coding RNA of ~19-25 nucleotides long that post-transcriptionally regulate protein expression by targeting messenger RNAs for cleavage or translational repression. MiRNAs have been implicated in the initiation and progression of 160+ human diseases. Unique miRNA differential expression signatures can be used as a basis of discriminating against the presence or absence of human diseases. MiRNAs are therefore a promising and emerging class of disease biomarkers and therapeutic targets; however, the accurate detection of a specific miRNA has continued to be a challenging issue. Recently, mass spectrometry (MS) has seen remarkable technological advancements making it an attractive alternative to the conventional molecular biology miRNA characterization techniques. This study consistently documents the development of various analytical techniques aimed at characterization of miRNAs. The current literature in the field of miRNA is covered in chapter one. In chapter two, two new MS based concepts for detection of miRNA are introduced; a) the miRNA is captured using a specific complementary DNA probe, eluted and digested with specific endonuclease. The digested miRNA fragments are measured by MS resulting in a peak pattern that is dependent on the miRNA sequence i.e. an intrinsic mass signature and b) a unique mass signature is created by incorporating extra nucleotide(s) to the 3' end of miRNA and the extended miRNA is measured by using MS. The molecular mass of the extended miRNA, which is defined as extended mass signature, is expected to be different from the other miRNA within the same sample. These two approaches can improve the accuracy on qualitative MS identification of specific miRNA. To better understand miRNA function however, it is important to elucidate the nucleotide sequence of the miRNA. Chapter three of this study introduces a novel MS based assay for the sequencing of miRNA through chemical hydrolysis. In this study, by taking advantage of the mixing between a miRNA sample and an acidic MALDI matrix prior to the MALDI-TOF MS measurements, a unique yet simple and relatively cost-effective approach to generate miRNA sequencing ladders was developed. By using this method, 100% sequence coverage and accuracy in the sequencing of selected miRNAs were achieved. When many samples are involved, the data generated from miRNA measurements can be complex and manual data processing is tedious and challenging, as such, the spectral interpretation of mass spectrometric data can quickly turn out to be the bottleneck in miRNA analysis. The success of MS as a tool for analysis of miRNA will therefore strongly depend on the development of relevant computational software with the ability to properly interpret and analyze the large data. To meet this need, chapter four of this work explains the development of MicroRNA MultiTool, a computational software for the rapid interpretation of MS data containing human miRNA. Users can directly enter data obtained from mass spectrometric measurement in order to obtain the identify of miRNA, highly reducing the time needed to process data. The development of such analytical and bioinformatics tools will provide scientists with the opportunity to better understand miRNA functions and will be influential in propelling the breakthroughs of miRNA in clinical diagnostics and therapeutic fields