A General Approach for Haplotype Phasing across the Full Spectrum of Relatedness

Many existing cohorts contain a range of relatedness between genotyped individuals, either by design or by chance. Haplotype estimation in such cohorts is a central step in many downstream analyses. Using genotypes from six cohorts from isolated populations and two cohorts from non-isolated populations, we have investigated the performance of different phasing methods designed for nominally ‘unrelated’ individuals. We find that SHAPEIT2 produces much lower switch error rates in all cohorts compared to other methods, including those designed specifically for isolated populations. In particular, when large amounts of IBD sharing is present, SHAPEIT2 infers close to perfect haplotypes. Based on these results we have developed a general strategy for phasing cohorts with any level of implicit or explicit relatedness between individuals. First SHAPEIT2 is run ignoring all explicit family information. We then apply a novel HMM method (duoHMM) to combine the SHAPEIT2 haplotypes with any family information to infer the inheritance pattern of each meiosis at all sites across each chromosome. This allows the correction of switch errors, detection of recombination events and genotyping errors. We show that the method detects numbers of recombination events that align very well with expectations based on genetic maps, and that it infers far fewer spurious recombination events than Merlin. The method can also detect genotyping errors and infer recombination events in otherwise uninformative families, such as trios and duos. The detected recombination events can be used in association scans for recombination phenotypes. The method provides a simple and unified approach to haplotype estimation, that will be of interest to researchers in the fields of human, animal and plant genetics.     

A Simple and Rapid Extraction of High Molecular Weight Chromosomal DNA from Bacillus subtilis Protoplasts for Cosmid Cloning and Interspecific Transformation

After conversion of Bacillus subtilis vegetative cells to protoplasts, a simple and rapid method for extracting high-molecular-weight chromosomal DNA was devised with the inclusion of bovine serum albumin and phenol-chloroform treatments. The DNA sample thus prepared was the size of 100-450 kb and could be used for cosmid cloning and interspecific transformation.     

DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival, as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multiclone and multicolor mapping experiments do not generate additional information. Our pooling protocol, described here with examples from thyroid cancer research and PGD, accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as 3 to 4 days. (J Histochem Cytochem 57:587–597, 2009)     

A Simplified Approach for the Molecular Classification of Glioblastomas

Glioblastoma (GBM) is the most common malignant primary brain tumors in adults and exhibit striking aggressiveness. Although GBM constitute a single histological entity, they exhibit considerable variability in biological behavior, resulting in significant differences in terms of prognosis and response to treatment. In an attempt to better understand the biology of GBM, many groups have performed high-scale profiling studies based on gene or protein expression. These studies have revealed the existence of several GBM subtypes. Although there remains to be a clear consensus, two to four major subtypes have been identified. Interestingly, these different subtypes are associated with both differential prognoses and responses to therapy. In the present study, we investigated an alternative immunohistochemistry (IHC)-based approach to achieve a molecular classification for GBM. For this purpose, a cohort of 100 surgical GBM samples was retrospectively evaluated by immunohistochemical analysis of EGFR, PDGFRA and p53. The quantitative analysis of these immunostainings allowed us to identify the following two GBM subtypes: the “Classical-like” (CL) subtype, characterized by EGFR-positive and p53- and PDGFRA-negative staining and the “Proneural-like” (PNL) subtype, characterized by p53- and/or PDGFRA-positive staining. This classification represents an independent prognostic factor in terms of overall survival compared to age, extent of resection and adjuvant treatment, with a significantly longer survival associated with the PNL subtype. Moreover, these two GBM subtypes exhibited different responses to chemotherapy. The addition of temozolomide to conventional radiotherapy significantly improved the survival of patients belonging to the CL subtype, but it did not affect the survival of patients belonging to the PNL subtype. We have thus shown that it is possible to differentiate between different clinically relevant subtypes of GBM by using IHC-based profiling, a method that is advantageous in its ease of daily implementation and in large-scale clinical application.     

Rapid Identification of Chromosomal Abnormalities in Human Neoplasia

Recent advances in banding techniques have led to the belief that certain chromosomal defects are consistently associated with specific types of human neoplasia. Based on the GTG technique, it has been suggested that the malignant cells of most neoplasias show chromosomal abnormalities (Yunis et al. 1983). From this recent publication of Yunis it appears that the majority of bands involved in carcinogenesis are G-negative, i.e., do not stain by the GTG technique, and it is therefore difficult to localize the breakpoints. In some of our recent publications we emphasized the importance of the RFA technique (Verma and Lubs 1975), which stains Giemsa-negative bands darkly, thus providing precise identification of chromosomal abnormalities (Verma and Dosik 1976). However, this technique cannot be applied until the slides have aged for at least 7 days. Therefore, we are reporting an alternative procedure using BrdU which provides “reverse” banding immediately when the slides are stained with acridness orange and examined with a fluorescence microscope.     

染色体末端微小结构异常的分子细胞遗传检测

为检出易于被忽略的染色体末端微小结构异常,为生育提供指导,选取特异性7号全染色体探针,X染色体长臂探针和7q亚端粒（7q36→qter)探针,用荧光原位杂交（fluorescence in situ hybridzation,FISH)结合G显带技术分析2个病例,其中病例1有不良妊娠史并疑有末端微小易位,病例2在G显带水平已发现为X和7号染色体易位的卵巢早衰患者,结果表明,FISH确诊病例1为染色体末端的隐匿易位,病例2的易位断点得到精确定位,它不在7q36而在7q末端。应用特异性染色体探针及亚端粒探针,通过FISH技术可以确诊染色体末端区域的微小结构异常,在临床遗传学中有广泛的应用,是遗传咨询和生育指导的有效工具之一。     

RFMix: A Discriminative Modeling Approach for Rapid and Robust Local-Ancestry Inference

Local-ancestry inference is an important step in the genetic analysis of fully sequenced human genomes. Current methods can only detect continental-level ancestry (i.e., European versus African versus Asian) accurately even when using millions of markers. Here, we present RFMix, a powerful discriminative modeling approach that is faster (∼30×) and more accurate than existing methods. We accomplish this by using a conditional random field parameterized by random forests trained on reference panels. RFMix is capable of learning from the admixed samples themselves to boost performance and autocorrect phasing errors. RFMix shows high sensitivity and specificity in simulated Hispanics/Latinos and African Americans and admixed Europeans, Africans, and Asians. Finally, we demonstrate that African Americans in HapMap contain modest (but nonzero) levels of Native American ancestry (∼0.4%).     

A Flexible Nanoarray Approach for the Assembly and Probing of Molecular Complexes

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers’ rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.     

Rapid Concentration and Molecular Enrichment Approach for Sensitive Detection of Escherichia coli and Shigella Species in Potable Water Samples

In this work, we used a rapid, simple, and efficient concentration-and-recovery procedure combined with a DNA enrichment method (dubbed CRENAME [concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment]), that we coupled to an Escherichia coli/Shigella-specific real-time PCR (rtPCR) assay targeting the tuf gene, to sensitively detect E. coli/Shigella in water. This integrated method was compared to U.S. Environmental Protection Agency (EPA) culture-based Method 1604 on MI agar in terms of analytical specificity, ubiquity, detection limit, and rapidity. None of the 179 non-E. coli/Shigella strains tested was detected by both methods, with the exception of Escherichia fergusonii, which was detected by the CRENAME procedure combined with the E. coli/Shigella-specific rtPCR assay (CRENAME + E. coli rtPCR). DNA from all 90 E. coli/Shigella strains tested was amplified by the CRENAME + E. coli rtPCR, whereas the MI agar method had limited ubiquity and detected only 65 (72.2%) of the 90 strains tested. In less than 5 h, the CRENAME + E. coli rtPCR method detected 1.8 E. coli/Shigella CFU whereas the MI agar method detected 1.2 CFU/100 ml of water in 24 h (95% confidence). Consequently, the CRENAME method provides an easy and efficient approach to detect as little as one Gram-negative E. coli/Shigella cell present in a 100-ml potable water sample. Coupled with an E. coli/Shigella-specific rtPCR assay, the entire molecular procedure is comparable to U.S. EPA Method 1604 on MI agar in terms of analytical specificity and detection limit but provides significant advantages in terms of speed and ubiquity.     

A Low Complexity Rapid Molecular Method for Detection of Clostridium difficile in Stool

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB ⁺, n =  111; tcdB ⁻, n  = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours.In a further study (tcdB ⁺, n =  47; tcdB ⁻, n  = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB ⁺, n =  25; tcdB ⁻, n  = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.     

Phasing statistics for alpha helical membrane protein structures

In this report we highlight the latest trends in phasing methods used to solve alpha helical membrane protein structures and analyze the use of heavy atom metals for the purpose of experimental phasing. Our results reveal that molecular replacement is emerging as the most successful method for phasing alpha helical membrane proteins, with the notable exception of the transporter family, where experimentally derived phase information still remains the most effective method. To facilitate selection of heavy atoms salts for experimental phasing an analysis of these was undertaken and indicates that organic mercury salts are still the most successful heavy atoms reagents. Interestingly the use of seleno‐l ‐methionine incorporated protein has increased since earlier studies into membrane protein phasing, so too the use of SAD and MAD as techniques for phase determination. Taken together this study provides a brief snapshot of phasing methods for alpha helical membrane proteins and suggests possible routes for heavy atom selection and phasing methods based on currently available data.     

Molecular Cloning and Chromosomal Localization of Human Salt-Tolerant Protein

We have isolated a novel human cDNA coding for human salt-tolerant protein (HSTP), that is a homologue of the rat salt-tolerant protein (STP) and may contribute to salt-induced hypertension by modulating renal cation transport. The nucleotide sequence (1988 bp) of the HSTP cDNA contains an open reading frame encoding a polypeptide comprising 545 amino acids, two residues fewer than the rat STP cDNA. The predicted amino acid sequence exhibits 92% identity to that of the rat protein. HSTP contains predicted coiled-coil domains and Src Homology 3 domain, and shows a high degree of identity to CIP4 (Cdc42 target protein) and human Trip 10 (thyroid-hormone receptor interacting protein). We have mapped the HSTPgene to human chromosome 19 by fluorescence in situhybridization. This revised version was published online in July 2006 with corrections to the Cover Date.